Methods for stabilizing biologically active agents encapsulated in biodegradable controlled-release polymers

ABSTRACT

Methods for reducing or inhibiting the irreversible inactivation of water-soluble biologically active agents in biodegradable polymeric delivery systems which are designed to release such agents over a prolonged period of time, such as PLGA delivery systems are provided. The method comprises preparing PLGA delivery systems whose microclimate, i.e. the pores where the active agent resides, uniformly or homogenously maintain a pH of between 3 and 9, preferably between 4 and 8, more preferably between 5 and 7.5 during biodegradation. Depending on the size of the delivery system, and the initial bulk permeability of the polymer, this result is achieved by (a) incorporating a water-soluble carrier into the delivery system, (b) incorporating a select basic additive (or antacid) into the delivery system, (c) incorporating both a water soluble carrier and a select basic additive into the delivery system, (d) adding a pore forming molecule for increasing the rate of release of low molecular weight monomers and oligomers into the delivery system, (e) using a PLGA polymer with reduced glycolide content, i.e. PLGA with from 100% to 75% lactide and 0 to 25% glycolide) (f) using a microencapsulation method that yields a more extensive pore-network, e.g. oil-in-oil emulsion-solvent extraction as opposed to water-in-oil-in water-solvent evaporation method, and (g) combinations thereof.

This application is a continuation of U.S. application Ser. No.10/700,107, filed on Nov. 3, 2003, the disclosure of which isincorporated herein by reference in its entirety, and U.S. applicationSer. No. 09/738,961 (now U.S. Pat. No. 6,743,446), filed on Dec. 15,2000, the disclosure of which is incorporated herein by reference in itsentirety. In addition, this application claims the benefit of U.S.Provisional Application No. 60/170,983, filed on Dec. 15, 1999, thedisclosure of which is incorporated herein by reference in its entirety.

This invention was made at least in part with government support underNational Institutes of Health Grant DE 12183. The government has certainrights in the invention.

BACKGROUND

Since the concept of protein or drug delivery from polymers was firstintroduced, research efforts have focused on developing polymerformulations that would be widely applicable for delivery ofbiologically active agents, such as proteins, peptides,oligonucleotides, DNA, low molecular weight drugs and vaccine antigens.Efforts to this end have intensified recently since hundreds ofrecombinant proteins and other biotechnological drugs and vaccineantigens are in the pipeline for FDA approval, and the current method ofprotein delivery generally requires injections on a daily basis.Frequent dosing is clinically undesirable due to patient discomfort,psychological distress, and poor compliance for administeringself-injections. To reduce injection frequency, peptide and proteindrugs are encapsulated in biodegradable polymers, which are processedinto a form that is easily administered through a syringe needle.Current preparations on the market for the delivery of small peptidescan reduce the frequency of injections to once every 1-3 monthsdepending on the size and dose of the polymer implant. This incubationtime, for which a large globular protein must remain encapsulated in thepolymer at physiological temperature, poses significant challenges toretain both the structural integrity and the biological activity of theprotein.

Two injectable polymer configurations are currently used to deliverpeptides and proteins: spherical particles on the micrometer scale(˜1-100 μm), which are commonly referred to as “microspheres,” andsingle cylindrical implants on the millimeter scale (˜0.8-1.5 mm indiameter), which we term “millicylinders.” Both configurations areprepared from the biocompatible copolymer class,poly(lactide-co-glycolide) (PLGA) commonly used in resorbable sutures,and each configuration has distinct advantages and disadvantages.

Once injected into the body, these polymer implants slowly release thebiologically active agents, thereby providing desirable levels of theagent over a prolonged period of time. Because of its safety, FDAapproval and biodegradability, the poly(lactide-co-glycolides) (PLGAs)are the most common polymer class used for preparing biodegradabledelivery systems for biologically active agents. Unfortunately, themicroenvironment in PLGA surrounding the encapsulated agent can becomehighly acidic, causing many of these agents to lose their biologicalactivity. Accordingly, it is desirable to modify the methods that arecurrently used to prepare polymeric delivery systems which liberateacids during biodegradation, such as PLGA, and to thereby produce apolymeric implant that is capable of releasing the biologically activeagent over a prolonged period of time and maintaining the stability ofthe biologically active agent that is retained in the delivery systemduring nonenzymatic hydrolysis, hereinafter referred to as“biodegradation” of such a system. Such methods would also be useful forpreparing implants that are made from polymers that contain acid thatslowly dissolves and lowers the pH of the microenvironment surroundingthe encapsulated agent

SUMMARY OF THE INVENTION

The present invention provides new methods for reducing or inhibitingthe irreversible inactivation of water-soluble biologically activeagents in biodegradable polymeric delivery systems which are designed torelease such agents over a prolonged period of time, such as PLGAdelivery systems. In accordance with the present invention, it has beendiscovered that, in many instances, the acids that are produced duringbiodegradation of PLGA can induce an irreversible inactivation orinstability of biologically active agents, such as for example proteins,drugs, oligonucleotides and vaccine antigens. It has also beendetermined that the addition of certain antacids, such as for exampleMgOH₂, to the system will not significantly reduce the acid-inducedinstability of the biologically active unless the polymer is prepared ina manner which results in the formation of an interconnected network ofpores within the polymer. It has also been discovered that theacid-induced instability of biologically active agents encapsulated inPLGA delivery can be inhibited or significantly reduced by preparingPLGA delivery systems whose microclimate, i.e. the pores where theactive agent resides, uniformly or homogenously maintain a pH of between3 and 9, preferably between 4 and 8, more preferably between 5 and 7.5during biodegradation. Depending on the size of the delivery system,i.e., the weight average particle diameter and the initial bulkpermeability of the polymer, this result is achieved by (a)incorporating a water-soluble carrier into the delivery system, (b)incorporating a select basic additive (or antacid) into the deliverysystem, (c) incorporating both a water soluble carrier and a selectbasic additive into the delivery system, (d) adding a pore formingmolecule for increasing the rate of release of low molecular weightmonomers and oligomers into the delivery system, (e) using a PLGApolymer with reduced glycolide content, i.e. PLGA with from 100% to 75%lactide and 0 to 25% glycolide) (f) using a microencapsulation methodthat yields a more extensive pore-network, e.g. oil-in-oilemulsion-solvent extraction as opposed to water-in-oil-in water-solventevaporation method, and (g) combinations thereof.

The present invention also relates to PLGA delivery systems prepared bythe present method. Such delivery systems have a low porosity (e.g.<50%) and a uniform morphology (e.g. spherical or cylindrical usuallywith smooth or uniformly rough surfaces, and when particulate, allparticles are similar in external and internal appearance under thescanning electron microscope. In addition, the PLGA delivery systems ofthe present invention have a low initial burst release (e.g. <50% of thedrug is released during the 1st hour of biodegradation) Mostimportantly, during biodegradation, the present PLGA delivery systemsmaintain a relatively homogenous microclimate pH greater than 3 and lessthan 9, preferably greater than 4 and less than 8, more preferablygreater than 5 and less than 7.5, so that less than 15% of the combinedreleased and residual encapsulated test protein bovine serum albuminforms nonconvalent, water-insoluble aggregates when incubated in aphysiological buffer solution for 4 weeks at 37° C.

In certain embodiments, the PLGA delivery system comprises bonemorphogenetic protein-2, vincristine sulfate, fibroblast growth factor,or tissue plasminogen activator.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of preparing PLGA deliverysystems which stabilize the soluble biologically active agents that areencapsulated therein. As used herein, the term stabilize refers to animprovement in the stability of the encapsulated agent, which isnecessary to approach or achieve a stable state. A stable biologicallyactive agent as used herein refers to a biologically active agent suchas a protein, peptide, oligonucleotide, low-molecular weight drug, orvaccine antigen that retains at least 80%, preferably 90%, of itsoriginal structure and/or biological activity during its release fromthe PLGA delivery system. During biodegradation of PLGA deliverysystems, soluble agents often undergo acid-induced irreversibleinstability. Such instability may result from noncovalent aggregation ofthe agent, peptide-bond hydrolysis, deamidation, isomerization, covalentaggregation, deformylation, depurination, etc. Each of theseacid-induced physical or chemical alterations can be monitored usingstandard techniques known in the art. For example, aggregation can bemonitored by loss of solubility, SDS-PAGE, and or size-exclusionchromatography.

The methods of the present invention also provide controlled releasePLGA delivery systems. As used herein, controlled release means therelease kinetics are engineered into the system such that the agent isreleased in a manner controlled by the system itself or itssurroundings, preferably the system itself. Such controlled releaserequires that the agent is not all released within a short period oftime, e.g., less than one hour, after injection or implantation of thesystem in a subject. Preferably the agent is released from the implantedsystem over a prolonged period of time, e.g. 3 days to 1 year. In somecases, the delivery system is designed to release the agent slowly andcontinuously over this prolonged period of time. In other instances thedelivery system is designed to release the agent in multiple phases.

Stabilization of the encapsulated agent is achieved by providing adelivery system whose microclimate, i.e. the pores where the activeagent resides, uniformly or homogeneously maintain a pH of greater than3 and less than 8, preferably greater than 4 and less than 8, morepreferably from 5 to 7.5 during biodegradation. To determine if themethod has provided a polymeric delivery system whose microclimatehomogenously maintains a pH of between 3 and 8, 1% w/w BSA is dispersedin the polymer solution during manufacture by the chosen method and theextent of aggregation of this protein is assayed after 4 weeks ofincubation of the polymeric delivery system in phosphate buffered salinewith 0.02% Tween 80 at 37° C. If the amount of residual BSA that hasformed water insoluble noncovalent aggregates (i.e., soluble in 6 Mguanidine hydrochloride or 6 M urea) is less than or equal to 15% of thetotal BSA in the prepared polymer dosage form, the method has produce apolymeric delivery system whose microclimate homogeneously maintains apH of between 3 and 8.

One method for preparing a delivery system which stabilizes the agentencapsulated therein during biodegradation comprises adding a poorlysoluble, mildly strong basic additive to a solution comprising thebiologically active agent and the polymer. Except for CaOH₂, the basicadditive has a solubility and basicity comparable to the solubility andbasicity of the compounds shown in Table 1 below.

TABLE 1 Solubility and basicity of basic salts. pH of saturated Additionof 100 Salts pK_(sp) ^(a) Solubility^(b) solution^(c) μl of 1 N HCI^(d)Ca(OH)₂ 5.26 1.11 × 10⁻² 12.40 12.20 CaCO₃ 8.42 6.17 × 10⁻⁵ 9.26 6.07Ca₃(PO₄)₂ 26.0 3.12 × 10⁻¹¹ 7.77 3.71 Mg(OH)₂ 10.74 1.66 × 10⁻⁴ 9.768.99 MgCO₃ 5.00 3.16 × 10⁻³ 9.75 9.01 Zn(OH)₂ 15.68 3.74 × 10⁻⁶ 8.855.86 ZnCO₃ 10.78 4.07 × 10⁻⁶ 7.34 5.36 Zn₃(PO₄)₂ 32.0 1.24 × 10⁻¹³ 6.821.53 ^(a)Lange's Handbook of Chemistry, Ed. John A. Dean, 11^(th)edition, 1973. ^(b)Solubility was calculated based on pK_(sp)values;^(c)pH was measured after excess basic salts were suspended in 10 ml ofwater and incubation was continued at 37° C. for 7 days; ^(d)pH wasmeasured after the acid was added to the above suspension and incubationwas continued at 37° C. for 3 days.

Suitable basic additives are magnesium carbonate, magnesium hydroxide,magnesium oxide, magnesium trisilicate, zinc carbonate, zinc hydroxide,zinc phosphate, aluminum hydroxide, basic aluminum carbonate,dihyroxyaluminum sodium carbonate, dihydroxyaluminum aminoacetate,ammonium phosphate, calcium phosphate, calcium hydroxide, magaldrate.Preferably, the polymer comprises from 50% to 100% lactide or lacticacid, which may be a D isomer, L-isomer, or a D,L-racemic mixture, andfrom 50% to 0% of a glycolide or glycolic acid. The polymer has aninherent viscosity of from 0.1 to 2.0 dl/g.

The polymer solution comprises from 0.1 to 20% of the biologicallyactive agent or a composition comprising the biologically active agentand a carrier. In those instances where the amount of biologicallyactive agent incorporated into the polymer solution is sufficient topromote formation of an interconnected network of pores, addition ofcarrier to the polymer solution is optional. In those cases where theamount of biologically active agent incorporated into the polymersolution is low (e.g., due to cost, toxicity, etc.), it is preferredthat a carrier be added. Examples of suitable carriers are albumin, gumarabic, gelatin, dextran, a water soluble amino acid, a monosaccharide,a disaccharide, and combinations thereof.

The polymer solution comprises from 0.5 to 20% of the basic additive. Inthose cases where the amount of basic additive dispersed in the solutionis low, i.e. from 0.5% to 3% w/w, it is preferred that the porosity ofthe polymeric delivery system be increased. Methods for increasing theporosity include adding a pore-forming agent to the polymer solution,increasing the amount of biologically active agent or the compositioncomprising the biologically active agent and carrier to a value of 5 to20% (w/w), or using a low concentration of polymer, e.g. 40-300 mg/ml ofpolymer in the organic solvent. In those cases where the polymerconcentration is high, e.g. 1200 mg/ml or the inherent viscosity ishigh, it is preferred that the polymer solution comprise from 3 to 20%by weight of the basic additive.

Another method of preparing biodegradable polymeric delivery systems forstabilizing the biologically active agents encapsulated therein involvesblending a pore-forming agent with a polymer which comprises from 50% to100% lactide or lactic acid and from 50% to 0% glycolide or glycolicacid. Examples of suitable pore-forming agents are polyethylene glycol(PEG) and water soluble poloxamers. Preferably, the pore-forming agenthas a molecular weight of from 500 to 30,000, more preferably from 4000to 10,000.

The methods of the present invention are suitable for preparing largedelivery systems having a weight average diameter of 5 to 500 mm,intermediate-sized delivery systems having a weight average diameter of100 to 5000 μm, and small delivery systems having a weight averagediameter of from 10 nm to 100 μm. The delivery systems of the presentinvention encompass spheres, including microspheres and nanospheres,cylinders, including millicylinders, and particles.

When aqueous soluble compounds are encapsulated in PLGA deliverysystems, they are typically distributed throughout the polymer. However,for many processes that are used to prepare PLGA delivery systems, thereis a large difference in content of the encapsulated compound at thesurface of the polymer relative to the bulk. This phenomenon, thepresence of acidic impurities in the polymer, and erosion events (e.g.,water uptake, acid-catalyzed polyester hydrolysis, sequestration oflow-molecular-weight acids, polymer permeability changes, pH-gradients,polymer glass transition changes, etc.) often result in a lowering ofmicroclimate pH in PLGAs.

Controlled-release systems for proteins and peptides usingpoly(lactide-co-glycolide) (PLGA) have been studied for more than onedecade. Although this type of biodegradable polymer has been successfulin delivery of small peptides such as LHRH analogues, the delivery oflarge globular proteins in PLGA has been limited because of theirreversible inactivation of these therapeutic agents prior to theirrelease in vivo. Previous work from our group has shown thatencapsulated bovine serum albumin (BSA) in PLGA systems forms insolublenon-covalent aggregates and is hydrolyzed after incubation in aphysiological buffer at 37° C. for 28 days. The acidic pH andintermediate water content existing in the polymer were implicated astwo major factors causing instability of the encapsulated protein, andthe BSA was stabilized by co-encapsulating poorly water-soluble basicinorganic salts such as Mg(OH)₂. The incorporation of the basic additivein the formulation was also successful in stabilizing therapeuticproteins such as recombinant human basic fibroblast growth factor andbone morphogenetic protein-2.

In this study, to further characterize the stabilization mechanism byco-encapsulation of Mg(OH)₂, the effect of basic additive type andcontent on protein stability and release kinetics in PLGA deliverydevices was studied. Since acid-induced inactivation pathways (e.g., atpH<3) are common for most proteins, BSA was selected as a model protein.BSA undergoes unfolding from its F to E form at pH 2.7, and formsnon-covalent aggregates in PLGA presumably due to this unfolding. Theinfluence of Mg(OH)₂ on the delivery system such as pH change in therelease medium, polymer degradation and water uptake kinetics was alsoexamined. In addition, the basicity of the salt as well as the loadingof base and protein were examined for their effects on BSA aggregation.

Our results confirm that below a critical loading of either basic saltor protein, both acidic and neutral pH regions in the polymer arepresent. Successful neutralization by the salt requires selection of theappropriate base as well as the appropriate combination of base andprotein loading, which allows the base to diffuse to all theprotein-containing pores and neutralize all the acidic regions in thepolymer.

Materials and Methods Chemicals

Poly(D,L-lactide-co-glycolide) 50/50 with inherent viscosity of 0.23,0.41, and 0.63 dl/g in hexafluoroisopropanol were purchased fromBirmingham Polymers, Inc. (Birmingham, Ala.). Bovine serum albumin(A-3059, Lot 32H0463) was purchased from Sigma Chemical Co. (St. Louis,Mo.). Poly(vinyl alcohol) (80% hydrolyzed with MW range of 8,000-9,000),Mg(OH)₂, Ca(OH)₂, and Ca₃(PO₄)₂ were obtained from Aldrich Chemical Co.(Milwaukee, Wis.). ZnCO₃ was from ICN Biopharmaceuticals Inc. (Aurora,Ohio). All these salts were fine powders (<5 μm) and were used asreceived.

Preparation of PLGA Cylindrical Implants

A solvent extrusion method similar to that used previously by our groupfor intraocular implants was used to prepare the PLGA cylinders with adiameter on the millimeter scale, which we term millicylinders. Briefly,a uniform suspension of sieved protein powder (<90 μm) with or withoutbasic salt in 50% (w/w) acetone-PLGA 50/50 solution was loaded in asyringe and extruded into a silicone tubing (I.D. 0.8 mm) at about 0.1ml/min. The solvent extruded suspension was dried at room temperaturefor 24 h and then dried in a vacuum oven at 45° C. for another 24 hbefore testing. The protein loading was calculated as the percentage ofamount of BSA versus the total weight of mixture (i.e., protein,polymer, and salt).

Evaluation of BSA Release from PLGA Implants

Release of protein was carried out in PBST (which consists of PBS (7.74mM Na₂HPO₄, 2.26 mM NaH₂O₄, 137 mM NaCl, and 3 mM KCl, pH 7.4), and0.02% w/v Tween® 80) at 37° C. under perfect sink conditions.Millicylinders (10×0.8 mm, 5-10 mg or microspheres (about 20 mg) wereplaced in 1 ml of the release medium and the medium was replaced at eachtime point. The protein content was determined by using Coornassie plusprotein assay reagent, which is also compatible with denaturing agents(e.g., 6 M urea) and reducing agents (e.g., 10 mM DTT).

Evaluation of BSA Stability within PLGA Implants

Protein stability was assessed by the percentage of water insolublenon-covalent BSA aggregates generated within the implants versus theinitial encapsulated protein. Protein stability within PLGA implants wasanalyzed as follows: First, millicylinders with a length of 1 cm wereincubated under 80% and 96% relative humidity (RH) at 37° C. for 21days. Then, the polymer was dissolved in acetone and centrifuged to spindown the protein. The remaining protein pellet was washed three timeswith acetone and then air-dried. The final protein pellet was analyzedas described in the section Analysis of the Protein Exacted From PLGAImplants. The protein remaining in PLGA implants after release in PBSTat 37° C. for 28 days was also extracted similarly and analyzed asabove.

Analysis of the Protein Extracted from PLGA Implants

The BSA pellet extracted from PLGA implants was first reconstituted inPBST and incubated at 37° C. overnight to determine the soluble proteinfraction remaining in the polymer. Any remaining aggregates werecollected by centrifugation again, and brought up in the denaturingsolvent (PBST/6 M urea/1 mM EDTA) and incubated at 37° C. for 30 min todissolve non-covalent bonded BSA aggregates. Then, any final undissolvedBSA aggregates were collected again and dissolved in the reducingsolvent (the denaturing solvent plus 10 mM DTT) to dissolve anydisulfide-bonded aggregates.

Protein Assay

For quantitation of soluble BSA, a modified Bradford assay was used asfollows: 10 μl of standard or sample in PBST was added to 250 μl ofCoomassle reagent/well on a 96-well plate and then the plate was read at595 nm using a Dynex MRX microplate reader (Dynex Technology, Inc.,Chantilly, Va.). The concentration range of the standard curve was 50 to1000 μg/ml. For quantitation of non-covalent and covalent BSAaggregates, the solvents used for preparation of standards and sampleswere 6 M urea and 6 M urea/10 mM DTT, respectively.

Measurement of Water Uptake in PLGA Millicylinders

After incubation either in PBST or under relative humidity at 37° C.,the millicylinders were blotted with tissue paper and weighedimmediately. They were then freeze-dried. The water uptake ofmillicylinders was calculated by:

Water uptake(%)=(W ₁ −W ₂)/W ₂×100%

Where W₁ and W₂ are the weights of the fully hydrated millicylinders andthe dried millicylinders, respectively.

Measurement of Molecular Weight of PLGA

Weight-averaged molecular weight (MW) of the degraded polymers wasmeasured by gel permeation chromatography (GPQ on a Styragel™ HR 5Ecolumn (7.8×300 mm, Waters, Milford, Mass.), which was performed on aHPLC system (Waters, Milford, Mass.) equipped with a refractive indexdetector (Hewlett Packard). The mobile phase was tetrahydrofuran with aflow rate of 1 ml/min. MW was calculated based on polystyrene standards(Polysciences Inc., PA) using Millenium Software Version 2.10.

SEM Image Analysis of PLGA Implants

Images of PLGA millicylinders were obtained by using a Philips XL30field emission gun scanning electron microscope (SEM). Samples werecoated with conductive-old palladium prior to the analysis.

pH Measurement of Saturated Basic Salts in Water

Basic salts (i.e., Mg(OH)₂, Ca(OH)₂, ZnCO₃ and Ca₃(PO₄)₂) in excess oftheir solubility were added to 5 ml of distilled water. The suspensionwas then incubated at 37° C. for 7 days. The pH of the supernatant wasdetermined with a Corning 430 pH meter (Corning Inc., N.Y.).

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the claims which are appended hereto.

Example 1 Effect of Mg(OH)₂ and Protein Loading on Stability inMillicylinders

Earlier work demonstrated that after an initial burst on the first day,BSA release from 15% BSA/millicylinders (0.63 dl/g PLGA 50/50) during 4weeks incubation in PBST at 37° C. is insignificant, and the remainingprotein mostly becomes water-insoluble non-covalent aggregates. It hasbeen shown that the BSA aggregation is mainly caused by acidicmicroclimate pH generated by polymer degradation and water uptake by thepolymer during incubation in PBST. It has also been found thatincorporation of 3% Mg(OH)₂ into 15% BSA/PLGA50/50 millicylinders canincrease BSA release from PLGA cylindrical implants and reduce BSAaggregation. Structural characterizations by using SDS-PAGE, IEF, CD,and fluorescence spectroscopy have confirmed that the structure of BSAfrom 3% Mg(OH)₂/15% BSA devices is mostly retained in a native form.

To examine the effect of Mg(OH)₂ content, the base was co-encapsulatedin 15% BSA/PLGA millicylinders as a function of base loading and the BSArelease. The study was carried out in PBST at 37° C. With the increasingMg(OH)₂ content from 0.5% to 6%, both BSA release rate and totalreleasable amount of protein increased. The residual BSA remaining inthese devices after the 4-week release interval was analyzed. In theabsence of Mg(OH)₂, most of the remaining protein became water insolubleaggregates, which were nearly completely soluble in the denaturingsolvent (i.e., non-covalent aggregates were formed). As the content ofMg(OH)₂ was increased, the amount of water-insoluble aggregatesdecreased. As Mg(OH)₂ content was raised to 6%, almost no aggregateswere formed within the device. For all the aggregates, an insignificantamount of covalent aggregates was observed in each polymer specimen.These results indicate that an increase in Mg(OH)₂ content even up to 6%does not generate an alkaline microclimate in the polymer duringrelease.

Stabilization of Encapsulated BSA in PLGA Implants by Mg(OH)₂ in thePresence of Moisture

The increased release rate of BSA in the presence of Mg(OH)₂ brings up apotential artifact when considering BSA stabilization by the salt. Ifthe salt only accelerates release, there may be insufficient time forBSA aggregates to form. To demonstrate the stabilization effect ofMg(OH)₂ in the implants with the same amount of encapsulated protein,15% BSA/PLGA implants with and without 3% Mg(OH)₂ were exposed to ahumid environment at 37° C., where all the protein remained inside thedevice during incubation and PLGA degradation occurred due to uptake ofwater vapor at 37° C. Two different humidities, 80% and 96% RH, wereselected since the salt may also affect water content of the protein,which also affects protein aggregation kinetics. After 3 weeksincubation, the remaining BSA was extracted from the polymer.

In the absence of the base, the same type of water-insolublenon-covalent BSA aggregates was observed among the remaining BSA, aspreviously reported during incubation in the release medium. With theincreasing relative humidity, the water content of the device increasedand the amount of the aggregates increased. Previous results have shownthat more than 60% of the initially encapsulated BSA formed non-covalentaggregates after 2 weeks release in PBST at 37° C. This indicates thatduring release the microclimate in the polymer may become more acidicdue, to the increased water uptake by the polymer. These results furtherdemonstrate that non-covalent BSA aggregation is caused by the acidicmicroclimate generated from PLGA degradation products.

In contrast, in the presence of 3% Mg(OH)₂ much less non-covalentaggregates were generated under both 80% and 96% RH conditions. Thisresult confirms that incorporation of Mg(OH)₂ in PLGA implants indeedcan inhibit non-covalent BSA aggregation in the absence of proteinrelease. Since the water uptake by the devices with or without the basewas similar, the stabilization effect of Mg(OH)₂ is most likely throughits neutralization of the acidic microclimate pH as the polymerdegrades. It has also been found previously that the amount of BSArelease from PLGA50/50 (0.64 dl/g) microspheres with or without Mg(OH)₂was almost identical after 28 days release, but the soluble proteinremaining in the polymer was significantly greater in the presence ofbase (i.e., 65% versus 17%). These results confirm the stabilizationeffect of the base and rule out the potential artifact due to the fasterrelease of the protein in the presence of the salt.

Characterization of Neutralization Effect of Mg(OH)₂ in PLGA Implants

To examine the mechanism of how Mg(OH)₂ improves BSA stability andenhances release from PLGA, the neutralization effect of Mg(OH)₂ in theacidic microclimate of PLGA millicylinders was examined. This effect wasconfirmed by changes in pH in the release medium and alteration of thePLGA degradation rate. During the release period from day 21 to 28, thepH of 500 μl release medium containing 5 mg, of 15% BSA/PLGAmillicylinder dropped to 3.5, while in the presence of 3% Mg(OH)₂ the pHwas still maintained around 7.0. The characterization of polymer MW byGPC also showed that degradation rate of the polymer in 15% BSA/PLGAmillicylinders was faster than that in the presence of Mg(OH)₂, whichsuggests that fewer acidic species were generated during release inthese Mg(OH)₂-containing millicylinders, consistent with the releasemedium pH data. Therefore, the Mg(OH)₂ inhibits the autocatalyticdegradation mechanism of PLGA. These results show that Mg(OH)₂ indeedneutralized the acidic microclimate, which is consistent with the resultreported by our group previously using a fluorescent probe. Thus, it isconcluded that Mg(OH)₂ stabilizes the encapsulated BSA throughneutralizing acidic species generated from PLGA degradation.

To explain the faster release profiles in the presence of Mg(OH)₂, thewater uptake kinetics of the millicylinders was characterized. Thepresence of 3% Mg(OH)₂ significantly increased the water uptake rate ofPLGA millicylinders. At 7 days, the total water content in 3%Mg(OH)₂/15% BSA/PLGA millicylinders was much higher than the polymerwithout Mg(OH)₂. This result suggests that the higher permeability isexpected in the millicylinders with Mg(OH)₂.

The reason that Mg(OH)₂ increases water uptake is likely due to changesin water activity within the PLGA millicylinders. Mg(OH)₂ increases themicroclimate pH in the polymer, which will cause the dissociation of theend groups (i.e., —COOH) with a pK_(a) of 3.83 for both glycolic andlactic acids of PLGA and ionization of the monomers/oligomers.Therefore, ionization of the polymer end groups and the increasedosmotic pressure will be the driving force for water molecules todiffuse into the polymer matrix, resulting in higher water content.

Effect of Protein Loading on BSA Release and Stability

Confocal micrographs of fluorescein-loaded PLGA microspheres withcoencapsulated Mg(OH)₂ and no protein, indicate a population of bothacidic and neutral pH pores in the polymer matrix. This pH heterogeneitysuggests that in order for BSA to be stabilized, the base must be ableto diffuse to the BSA-containing pores. Moreover, from control studieswe have observed that 15% protein loading is sufficient for BSA topercolate effectively throughout the polymer. For example, if the BSAloading without the base is increased to 20%, >90% of the protein isrelease in 1 day. Therefore, a decrease in the percolation of BSAparticles in the polymer with basic salt would be expected to cause arise in BSA aggregation, corresponding to increased exposure of BSA toacidic pores.

To test this hypothesis, BSA loading was decreased to reduce itspercolation, and the BSA release was studied. As expected, as theloading was decreased. an increase in BSA aggregation was observed. AsBSA loading was decreased there was also a corresponding decrease in therelease rate. A similar phenomenon was observed for the stabilization ofBSA in PLGA50/50 microspheres at a similar BSA loading (4%) to the 5%BSA/PLGA millicylinders.

Example 2 Effect of MgCO₃ on Protein Stability in PLGA Millicylinders

A higher soluble base, MgCO₃, stabilized BSA much better than theMg(OH)₂, even though both bases neutralize acidity in a saturatedsolution to the same extent.

Example 3 Effect of ZnCO₃ and Ca(PO₄)₂ and Ca(OH)₂ on Protein Stabilityin PLGA Millicylinders

To examine the effect of the basic salts with different alkalinity onthe stability and release of BSA encapsulated in the polymer, onerelatively strong basic salt, Ca(OH)₂, was chosen and two otherrelatively weak basic salts, ZnCO₃ and Ca₃(PO₄)₂, were chosen andexamined as to whether similar stability and release profiles could beachieved in 15% BSA/PLGA millicylinders, as was demonstrated with theuse of Mg(OH)₂ in Example 1 above. The solubilities and pH of thesesalts are shown in Table 1.

TABLE 2 Effect of basic salts on BSA aggregation in 15% BSA/PLGA (0.63dl/g) Millicylinders after 2-week release study in PBST at 37° C.(average ± SEM, n = 3) Salts Ca(OH)₂ ZnCO₃ Ca₃(PO₄)₂ % 0.5 3.0 0.5 3.00.5 3.0 Soluble BSA^(a), % 51 ± 4 13 ± 1 36 ± 1 30 ± 1 44 ± 2 52 ± 1 Non-covalent aggregate^(b), % 10 ± 1  3.9 ± 0.1 30 ± 4 10 ± 1 30 ± 2 8.4± 0.3 Covalent aggregate^(c), % n.d.^(d) 11 ± 1 n.d.  1.8 ± 0.1 n.d. 1.0± 0.1 ^(a)Soluble in PBST; ^(b)Soluble in PBST containing 6 M urea and 1mM EDTA; ^(c)Soluble in PBST containing 6 M urea, 1 mM EDTA and 10 mMDTT; ^(d)n.d. — not detectable

With the increasing content of ZnCO₃, Ca(OH)₂, or Ca₃(PO₄)₂, both therelease rate and total releasable amount of BSA increase, which is quitesimilar to the effect of Mg(OH)₂. Analysis of the residual BSA in thesedevices is discussed next. For ZnCO₃ and Ca₃(PO₄)₂, similar to Mg(OH)₂,the total amount of water insoluble aggregates decreased with theincreasing salt content. For Ca(OH)₂, only 0.5% of Ca(OH)₂ of theloading was required to attain a similar inhibition of BSA aggregationas attained with 3% of the weak bases. However, when the loading wasraised to 3%, a significant amount of covalent bonded aggregates of BSAformed, which suggests that the microclimate pH in the presence ofCa(OH)₂ becomes more alkaline than with the weakly basic salts. Comparedto a pH of 9.97 for saturated Mg(OH)₂ solution, the pH of a saturatedCa(OH)₂ solution was found to be 12.4. As pH becomes alkaline, the freethiol group of Cys residues ionizes to become the more reactive thiolateand readily catalyzes disulfide bonded BSA aggregates viathiolate-disulfide interchange. For ZnCO₃ and Ca₃(PO₄)₂, the pH of theirsaturated solution was found to be 7.34 and 7.77, respectively, whichindicates that both species are very weak bases. Therefore, compared toMg(OH)₂, after only two weeks incubation, a larger amount ofnon-covalent aggregates in PLGA millicylinders with ZnCO₃ or Ca₃(PO₄)₂were observed and no detectable amount of covalent aggregates wereformed (Table 2).

The reason that the BSA release was relatively faster in the presence ofZnCO₃ than Ca₃(PO₄)₂ may be explained as follows: Since ZnCO₃ will reactwith protons generated from PLGA degradation to form a weak acid H₂CO₃(pKa_(i)=6.35), and while for Ca₃(PO₄)₂ a strong acid H₃PO₄(pKa_(i)=2.16) will be produced, it is expected that the microclimate inthe millicylinders with Ca₃(PO₄)₂ will be more acidic than that withZnCO₃. This was confirmed by the following experimental data simulatingthe reaction of acidic species and basic: salt occurring in the polymer:when 100 μl of 1 N HCl was added to a saturated ZnCO₃ solutioncontaining excess of salt, the pH dropped to 5.36, whereas the sameamount of HCl added to a saturated Ca₃(PO₄)₂ solution caused the pH todrop to 3.71. The difference of the neutralization effect from the saltsis also reflected in the different water contents of their PLGA devices.The 3% ZnCO₃/15% BSA/PLGA millicylinders had a water content of 168±5%(n=3) after 2-week release compared to 81±1% (n=3) in the 3%Ca₃(PO₄)₂/15% BSA/PLGA millicylinders. which suggests that ZnCO₃ shouldraise the microclimate pH in the polymer greater than does Ca₃(PO₄)₂.These results show that the homogeneity of the microclimate pH insidePLGA implants can be controlled by selecting certain types of basicsalts, which suggests a potential approach to optimize the stability ofencapsulated pharmaceuticals in PLGA including, therapeutic proteins.

Overall, as seen in this study, although adding certain percentage ofbasic salts to BSA/PLGA devices can reduce the aggregation and enhancethe release, higher content of salts results in shorter release durationwhile lower content of salts cannot eliminate aggregation. Therefore, itmay desirable to add other excipients, such as sucrose to the deliverysystems which contain high levels of salts in order to increase releaseduration. In a recent study, we found that encapsulation of sucrose into15% BSA/3% Mg(OH)₂/PLGA can also minimize the amount of aggregatesformed during release but slow down the release rate.

Example 4 Investigation of Protein Release and Stability in PLGAMicrospheres

The purpose of the work in this example is to 1) investigate the proteinrelease and stability in PLGA microspheres (which are prepared by themethods different from cylindrical implants and have smaller geometry;i.e., 10-100 μm), and 2) test whether the stabilization approachdeveloped for the cylindrical implants was applicable for proteindelivery from this more challenging PLGA system. To achieve theseobjectives, BSA was chosen as a model protein and its release andstability was studied in PLGA microspheres. First, the standardwater-in-oil-in-water (W/O/W) double emulsion and solvent evaporationmethod was used to encapsulate BSA into PLGA microspheres. As expected,the same BSA aggregation mechanism and incomplete releasecharacteristics were observed during in vitro release as for thecylindrical implants. When basic salts (e.g. ZnCO₃, Mg(OH)₂, and MgCO₃)were co-encapsulated into the microspheres made of high MW PLGA50/50(0.64 dl/g), the BSA aggregation rate was reduced but no significantamount of protein was released over one month. However, continuousprotein release was achieved when the microspheres were prepared fromlow MW polymer (0.20 dl/g) in the presence of basic salts. In addition,protein release and stability in PLGA microspheres prepared by anoil-in-oil (O/O) emulsion encapsulation method were also studied. It wasalso observed that in the presence of basic salts (e.g., Mg(OH)₂ andZnCO₃), continuous protein release from O/O microspheres was achievedand less encapsulated protein became aggregates compared to withoutbasic salts. This study further confirms the effectiveness of utilizingbasic salts to stabilize the encapsulated proteins and to control theirrelease from PLGA microspheres.

Poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA)polymers were purchased from Birmingham Polymers, Inc. (Birmingham,Ala.). PLGA50/50 polymers with inherent viscosity of 0.20, 0.64 and 0.70dl/g were used, and PLGA75/25, PLGA85/15 and PLA had inherent viscosityof 0.58, 0.66 and 0.69 dl/g, respectively. Bovine serum albumin (A-3059,Lot 32H0463), Span 85 surfactant, and cotton seed oil were from SigmaChemical Co. (St. Louis, Mo.). Petroleum ether with 50-110° C. boilingrange was obtained from J. T. Baker (Phillipsburg, N.J.). Actonitrileand acetone (analytical grade) were from Fisher Scientific Co.(Pittsburgh, Pa.). Poly(vinyl alcohol) (PVA) and fine Mg(OH)₂ powder (<5μm) were from Aldrich Chemical Co. (Milwaukee, Wis.) and ZnCO₃ powderwas from ICN Pharmaceuticals Inc. (Aurora, Ohio). All other chemicalswere of analytical grade or purer and purchased from commercialsuppliers.

For preparation of PLGA50/50 (0.64 dl/g) microspheres, 100 μl of BSAsolution (150 mg/ml or 300 mg/ml) in 10 mM phosphate buffer (pH 7.4) wasfirst added to 1 ml of 30% w/v PLGA-CH₂—CL₂ solution with or withoutbasic salts. Then, the mixture was homogenized at 10,000 rpm using ahomogenizer (Model IQ², VirTis Co., Gardiner, N.Y.) for 1 min in an icebath. The formed W/O emulsion was immediately added to 1 ml of 2% w/vpolyvinyl alcohol (PVA) aqueous solution and the mixture was vortexedfor 20 s to form a W/O/W emulsion. The double emulsion was immediatelytransferred to 100 ml of 0.5% w/v PVA aqueous solution under stirring ata constant rate. The microspheres were stirred continuously for 3 h atroom temperature. The hardened microspheres were collected bycentrifugation and washed with ice-cold water 3 times. Finally, themicrospheres were lyophilized for 24 h to get the final dry product witha Labcono FreeZone® 6 Liter Freeze Dry System (Kansas City, Mo.).

For preparation of PLGA50/50 (0.20 dl/g) microspheres, all the materialsand procedures were the same except that 70% w/v polymer concentrationwas used instead of 30%.

To prepare PLGA microspheres by the oil-in-oil (O/O) emulsionmethod/solvent extraction method, BSA particles (directly ground fromthe lyophilized powder and sieved to <45 μm) were added to a polymersolution in 1 ml of facetonitrile. The suspension was homogenized at15,000 rpm with a homogenizer (Model IQ², Virtis Co., Gardiner, N.Y.)for 3 min on an ice bath, and then slowly was added dropwise to a 100 mlof cotton seed oil (Sigma Chemical Co.) containing 1.6 grams of Span 85under stirring at 700 rpm. The formed O/O emulsion was continued to stirunder ambient conditions for 5 hr. Thereafter, 100 ml of petroleum etherwas added and stirring was continued for another 15 min. Themicrospheres were then collected by filtration through a 0.45 μmmembrane filter (Gelman Sciences) and then lyophilized at roomtemperature for 2 days.

To prepare BSA/PLGA microspheres by the W/O/W emulusion method, toreduce the burst effect, generally the volume ratio of the internalphase (protein solution) to the external phase (polymer solution) shouldbe below 1/10 and higher polymer concentration should be used [Cleland1997]. Therefore, in this study, the ratio of 1/10 and the PLGA50/50(0.64 dl/g) concentration of 300 mg/ml (700 mg/ml was used for PLGA50/50(0.20 dl/g)) for all the preparations, which resulted in highencapsulation efficiency for these preparations (i.e., >80%). By SEM,PLGA microspheres prepared by this method appeared mostly spherical withvery smooth surfaces and their size range was between 60 and 70 μm.

The in vitro release and stability of BSA encapsulated in W/O/W PLGAmicrospheres was examined. More specifically, the in vitro releaseprofiles of 4% and 8% BSA loaded PLGA50/50 (0.64 dl/g) microspheres fromthe W/O/W preparations were studied. The burst effect increased with theBSA loading likely due to more percolation protein clusters formedacross the microsphere diameter. However, over the 28-day release, both4% and 8% BSA preparations did not release significant amount of proteinfrom the polymer after the first day initial burst. This incompleterelease phenomenon is quite similar to results published in theliterature.

At the end of release study, after removing the polymer with acetone,the remaining BSA in the polymer was also found to be partiallyinsoluble in PBST buffer (Table 2). However, all these aggregates weresoluble in 6 M urea, which indicates that encapsulated BSA also formednon-covalent aggregates during release from PLGA microspheres. Thisresult shows that formation of non-covalent BSA aggregates is a commonphenomenon in both PLGA millicylinders and microspheres, which suggeststhat the microclimate in the PLGA microspheres during release may alsobecome very acidic and could be equivalent to the pH in the PLGAmillicylinders (pH<3).

TABLE 3 Aggregation of encapsulated BSA in PLGA50/50 (0.64 dl/g)microspheres over 28-day release in PBST at 37° C. (Average ± SEM, n =3) BSA loading, Released, Soluble residue, Insoluble residue, % % % % 44.4 ± 0.1 17 ± 2 68 ± 6 8 23 ± 1  45 ± 1 25 ± 3

The incomplete release and non-covalent BSA aggregation also occurred inthe PLGA50/50 microspheres made of lower MW polymer (0.20 dl/g). Nosignificant amount of protein was released from 3% BSA/PLGA50/50 (0.20dl/g) microspheres over 51 days release, and the remaining BSA mostlybecame water-insoluble non-covalent aggregates. This result indicatesthat non-covalent aggregation of encapsulated BSA in PLGA is a commoninstability pathway no matter whether high MW or low MW polymer is used.

The effect of basic salts on BSA release and stability in W/O/Wmicrospheres was studied. Since non-covalent aggregation of encapsulatedBSA indicates existence of the acidic microclimate in the PLGAmicrospheres during release, to examine whether basic salts such asMg(OH)₂ could also be used to inhibit BSA aggregation in PLGAmicrospheres, Mg(OH)₂ was co-encapsulated into 4% BSA/PLGA50/50 (0.64dl/g) microspheres. The in vitro release and stability over 28-dayrelease in PBST medium at 37° C. were studied. The cumulative BSArelease over 28 days did not increase significantly with Mg(OH)₂content, which is different from that of PLGA millicylinders. However,the BSA aggregates in the remaining protein from the polymer decreasesignificantly with increasing Mg(OH)₂ content from 0.5% to 3%, whichconfirms that non-covalent BSA aggregation can also be inhibited byneutralizing the acidic microclimate in PLGA microspheres with Mg(OH)₂.

In most protein delivery applications, continuous release profiles formPLGA microspheres are desirable. To examine whether increasing BSAloading can achieve continuous release in the presence of 3% Mg(OH)₂, 8%BSA loaded PLGA microspheres were prepared. Their in vitro releaseprofiles were studied. Similar to that in the absence of Mg(OH)₂,increasing BSA loading only increased the initial burst but did notachieve continuous release effect in the presence to 3% Mg(OH)₂.

To compare the effect of other basic salts on the release and stabilityof BSA encapsulated in PLGA microspheres by the W/O/W emulsionpreparation, MgCO₃, ZnCO₃, and CaCO₃ were co-encapsulated into 4%BSA/PLGA50/50 (0.64 dl/g) microspheres. Their in vitro release profileswere studied. Here, MgCO₃ allowed BSA to continuously release up to 10%over 28 days and all other basic salts did not significantly increasethe release of BSA from PLGA microspheres (<5%) after the first dayburst. This may be caused by higher water uptake in the presence ofMgCO₃ due to its higher solubility. Upon 28 days release, the remainingBSA was extracted and the aggregation results were noted. As expected,compared to that without basic salts (Table 3), all the basic saltsincreased the soluble residue and reduced the insoluble residue. Due tothe more alkaline property of Mg(OH)₂ and MgCO₃, the incorporation ofthese salts increased the soluble BSA residue more compared to theweaker bases (CaCO₃ or ZnCO₃), which further confirmed theneutralization effect of basic salts on the stability of encapsulatedBSA in PLGA microspheres.

TABLE 4 Effect of basic salts on BSA aggregation in W/O/W 4% BSA/PLGA50/50 (0.64 dl/g) microspheres over 28-day release in PBST at 37° C.(Average ± SEM, n = 3) Basic salts Released, % Soluble residue, %Insoluble residue, % 3% CaCO₃ 10 ± 1  33 ± 1 42 ± 1 3% ZnCO₃ 9.8 ± 0.146 ± 1 33 ± 1 3% Mg(OH)₂ 6.9 ± 0.2 65 ± 2 26 ± 1 3% MgCO₃ 17 ± 2  59 ± 113 ± 2

Although the incorporation of the basic salts indeed inhibited theprotein aggregation, it did not significantly increase the proteinrelease from microspheres made of high MW polymer (0.64 dl/g). Toexamine whether these basic salts can increase protein release frommicrospheres made of lower MW polymer, they were co-encapsulated into 3%BSA/PLGA50/50 (i.v.=0.20 dl/g) microspheres. The release profiles werestudied. In the absence of basic salts, a very small amount ofencapsulated BSA was released over 51 days. In contrast, in the presenceof 3% Mg(OH)₂, ZnCO₃, or Mg(OH)₂, the BSA release rate increased,especially for 3% MgCO₃. Therefore, for the microspheres with lower MWPLGA, co-encapsulation of basic salts can lead to continuous proteinrelease from the polymer. This may result from higher permeability ofthe lower MW polymer matrix due to its more hydrophilicity and lowerglass transition temperature. At the end of the release study, thecomposition of the remaining BSA in the microspheres was analyzed andlisted in Table 4. Without basic salts, the total detectable BSA amountwas below 50% of initially encapsulated protein based on thepre-determined BSA loading, which may be caused by extensive proteinhydrolysis due to exposure to acidic microclimate for 51 days. Theco-encapsulated basic salts actually increased the detectable amount ofBSA although a significant amount of non-covalent aggregates were alsogenerated even in the presence of 3% Mg(OH)₂.

TABLE 5 Effect of basic salts on BSA aggregation in 3% BSA/PLGA50/50(0.20 dl/g) microspheres over 51-day release in PBST at 37° C. (Average± SEM, n = 3) Basic salts Released, % Soluble residue, % Insolubleresidue, % No salts 16 ± 2 0.9 ± 0.1 24 ± 3 3% ZnCO₃ 41 ± 1 0.5 ± 0.1 22± 1 3% Mg(OH)₂ 37 ± 2 2.1 ± 0.1 30 ± 2 3% MgCO₃ 68 ± 2 24 ± 1   1.5 ±0.2

To understand how each basic salt affected BSA aggregation in the lowerMW PLGA microspheres, the pH changes in the release medium wereexamined. ZnCO₃ did not show any obvious neutralization effect. The factthat ZnCO₃ increased BSA release may be due to: 1) ionized polymer endgroups (—COOH) caused by localized pH neutralization, 2) enhanced wateruptake due to the formation of soluble Zn²⁺ and HCO₃ ⁻ ions in theacidic pH. In the presence of 3% Mg(OH)₂ or MgCO₃, initially the pH ofthe release medium was higher than without basic salts. However, after35 days, the pH also became acidic (<4.5) even in the presence ofMg(OH)₂, which may be due to the depletion of the basic salts caused byextensive polymer erosion. In the case of MgCO₃, due to its highersolubility in water than Mg(OH)₂, more than 50% of initiallyencapsulated BSA was released, so the polymer matrix should be morepermeable to many acidic polymer degradation products, which is expectedto have less acidic microclimate and result in fewer BSA aggregates.While in the case of Mg(OH)₂, most of the protein still remained in thepolymer end and therefore, the microspheres should have a lowerpermeability. After the base polymer is depleted, the acidic specieswill be accumulated, which causes aggregation. Therefore, selection ofspecific basic salt type and content is critical to pH control, whichappears to be necessary for stabilization and controlled release ofproteins encapsulated in PLGA systems.

Example 5 Preparation and Study of BSA/PLGA Microspheres Prepared by O/OEmulsion Method

Encapsulated BSA formed non-covalent aggregates in W/O/W PLGAmicrospheres during release, which can be inhibited by co-encapsulationof basic salts such as Mg(OH)₂ and MgCO₃. To test whether the stabilityof encapsulated BSA in PLGA microspheres during release is encapsulationmethod dependent, BSA was encapsulated into PLGA microspheres by anothercommonly used anhydrous encapsulation method—the oil-in-oilemulsion/solvent extraction method. For O/O preparations, a severe bursteffect and poor emulsion stability were two major problems. In order tosolve these problems, several formulation variables may be controlled,including polymer composition (lactide/glycolide ratio), polymerconcentration, and protein loading. Therefore, the first objective ofthis study was to prepare spherical PLGA microspheres with a low bursteffect by adjusting these variables.

The effect of polymer lactide/glycolide ratio on preparation and initialburst of these microspheres was studied. Since it is known that PLGAmicrospheres made of higher MW polymer have lower burst effect, a seriesof PLGA polymers with similar inherent viscosity (0.58-0.70 dl/g) butdifferent lactide/glycolide (LA/GA) ratio (50/50, 75/25, 85/15 and100/0) were chosen. Using a polymer concentration of 300 mg/ml, 5%loaded BSA microspheres were prepared. In all the preparations, theemulsion was very stable and could be observed in the SEM. All thesepreparations resulted in very spherical microspheres with smoothsurfaces. Most of the microspheres have the average size range from 80to 100 μm. Also this preparation method has a very high encapsulationefficiency (>94%).

The effect of polymer concentration on preparation and initial burst wasstudied. In O/O emulsion preparations, low polymer concentrations (below100 mg/ml) were usually used in order to form spherical microspheres. Toexamine how the polymer concentration affects the emulsion in thispreparation method, 100, 200, 300, 400 mg/ml of PLGA85/15 (0.66) weretested in the presence of 5% BSA particles. For polymer concentrationsfrom 100 to 300 mg/ml, the emulsions were very easily formed andspherical particles with smooth surfaces were obtained. The average sizeof microspheres increased from 63 to 93 μm with increasing polymerconcentration from 100 to 300 mg/ml. In this polymer concentrationrange, all the preparations had very high encapsulation efficiency(>91%; Table 6). For 400 mg/ml, however, the emulsion could not beformed because the polymer solution was too viscous.

TABLE 6 Effect of polymer concentration on preparations of 5%BSA/PLGA85/15 (0.66 dl/g) microspheres and the initial burst. PolymerConc. Size BSA loading Enapsulation 1^(st) day release (mg/ml) (μm)^(a)(%)^(b) efficiency (%)^(c) (%)^(b) 100 63 ± 15 4.56 ± 0.22 91 68 ± 1 20079 ± 30 4.76 ± 0.11 95 48 ± 1 300 92 ± 22 4.80 ± 0.03 96 22 ± 1^(a)average ± SD, n = 50; ^(b)average ± SEM, n = 3; ^(c)encapsulationefficiency = measured loading/calculated loading × 100%.

No obvious protein particles were found on the surface, which indicatesmost of the protein particles were encapsulated into the microspheres.However, the burst effect increased dramatically with decreasing thepolymer concentration, which may be caused by more porous structure ofPLGA microspheres with low polymer concentration.

To test the effect of BSA loading on preparation of PLGA microspheres,5, 10, 15% BSA loaded PLGA85/15 microspheres were prepared at a polymerconcentration of 300 mg/ml. It was found that most microspheres werevery spherical and had very smooth surfaces. No observable proteinparticles were absorbed on the surface, which indicates that mostprotein was encapsulated into the polymer. All the preparations alsoshowed high encapsulation efficiency (>90%; Table 7). As expected, withthe increasing BSA loading the initial burst increases, due to theformation of more percolation clusters of protein particles across themicrosphere diameter.

TABLE 7 Effect of BSA loading on preparations of PLGA85/15 (0.66 dl/g)microspheres and the initial burst. BSA loading Size BSA loadingEnapsulation 1^(st) day release (%) (μm)^(a) (%)^(b) efficiency (%)^(c)(%)^(b) 5 92 ± 22 4.80 ± 0.03 96 22 ± 1 10 94 ± 27 9.59 ± 0.21 96 46 ± 115 102 ± 30  13.5 ± 0.12 90 67 ± 2 ^(a)average ± SD, n = 50; ^(b)average± SEM, n = 3; ^(c)encapsulation efficiency = measured loading/calculatedloading × 100%.

The effect of formulation variables on BSA release and stability in O/Omicrospheres was studied. To test whether continuous protein release isachievable by adjusting the formulation variables by the O/O emulsionmethod, the in vitro release kinetics of microspheres prepared from PLGAwas examined as a function of LA/GA ratio, polymer concentration, andprotein loading. The results showed that, over the 35-day release,except for PLGA50/50, all these formulations released less than 10%protein from microspheres after the first day initial burst, whichsuggests that continuous protein release was not achievable by simplychanging these formulation variables. For PLGA50/50 microspheres, thesecond burst was likely caused by the known pulse of mass loss from thistype of polymer during erosion. The 50:50 LA/GA ratio results in fasterpolymer degradation compared to the other polymers of lower glycolidecontent.

At the end of release study, the remaining protein extracted from thepolymer was analyzed and the stability noted. The data showed that theLA/GA ratio of PLGA directly affected the stability of encapsulated BSAduring release. In the PLGA75/25 and PLA microspheres, very small amountof remaining BSA formed non-covalent aggregates, whereas in thePLGA50/50 microspheres most of remaining protein became water-insolubleaggregates. This result further confirms that the acidic microclimatecaused by PLGA degradation is the major source responsible for theformation of non-covalent BSA aggregate during release, becausePLGA50/20 degradation rate is faster than other polymers and therebymore acidic species will be generated over 35-day release. This isconfirmed by the measurement of pH change in the release medium. ForPLGA50/50 microspheres, the pH dropped significantly after 20 days.While for PLGA75/25 and PLA microspheres, the pH still remained atneutral pH over 35 days.

The possible cause for the increased BSA aggregation in PLGA85/15microspheres relative to PLGA75/25 and PLA is the presence of more lowMW species in PLGA85/15. The presence of monomers or oligomers can alsoproduce acidic microclimate even before polymer degradation occurs. Thishypothesis was confirmed by the initial pH drop (on day 1 and 4) in therelease medium containing PLGA85/15 microspheres, which is likely due torelease of these low MW acidic species into the release medium.

The acidic microclimate in the PLGA50/50 microspheres is also shown inthe degradation of the remaining soluble protein from the polymer after35 days incubation in PBST at 37° C. The data showed that the remainingsoluble BSA extracted from PLGA75/25, PLGA85/15, and PLA microspheresmostly still retained the same MW as the standard BSA, while the proteinfrom the PLGA50/50 microspheres contained a significant amount ofpeptide fragments. This result indicates that PLGA50/50 microspheres hada much more acidic microclimate than PLGA75/25, PLGA85/15 or PLAmicrospheres.

Example 6 Preparation and Study of rhBMP-2/PLGA Microspheres

To prepare the rhBMP-2/PLGA microspheres by the standardwater-in-oil-in-water emulsion method, one major difficulty is the lowaqueous solubility of rhBMP-2 at neutral pH, which prevents attainmentof a high loading of rhBMP-2 in PLGA microspheres. To overcome thisproblem, the first approach was to add heparin as a polyanion toincrease rhBMP-2 solubility at neutral pH. It was found that when theweight ratio of heparin/rhBMP-2 was above 4, rhBMP-2 remained solubleeven at 20 mg/ml in water. Therefore a heparin-rhBMP-2 complex(heparin/rhBMP-2 ratio˜7) was co-encapsulated with BSA into PLGAmicrospheres. However, the in vitro results showed that significantbleeding was observed surrounding the implants, which indicates thatheparin cased hemorrhage. The second approach was to decrease the pH inthe buffer to increase rhBMP-2 solubility. Since BSA is not stable atlow pH, obviously BSA is not a good candidate as a bulk excipient forrhBMP-2 delivery from PLGA microspheres. Although the salts present inrhBMP-2 lyophilized cake may cause a large burst effect due to theirhigh osmotic pressure, rhBMP-2 has a relatively lower solubilitycompared to these salts, suggesting that the burst effect may not occurfor rhBMP-2. Therefore, 400 μl of water was directly added to one vialof rhBMP-2 lyophilized cake to prepare a 20 mg/ml rhBMP-2 solutioncontaining a high concentration of salts at pH 4.5. Although rhBMP-2 isa relatively stable growth factor at an acidic pH below roomtemperature, rhBMP-2 may not survive when exposed to both bodytemperature and acidic microclimate caused by PLGA degradation (e.g.,pH<2). Therefore, based upon previous results, in this formulation 5%ZnCO₃ was also added as a neutralizing substance to prevent thegeneration of extremely low pH in the polymer during release. Meanwhile,ZnCO₃ also can increase the polymer water uptake and give proteincontinuous release from the device.

The SEM images of the microspheres prepared from different polymerconcentrations showed that the microspheres from lower polymerconcentration were more porous than those prepared from higher polymerconcentration. The polymer concentration also affected both theencapsulation efficiency and particle size. With increasing the polymerconcentration, both the encapsulation efficiency and average particlesize increased (Table 8) which may result from the increased viscosityof the polymer phase.

TABLE 8 Characteristics of rhBMP-2/PLGA50/50 delivery devices. rhBMP-2Encapsulation loading, %^(c) efficiency, %^(d) Device size Cylindrical0.25 ± 0.01 83 o = 0.32 cm, implants L - 0.4 cm Microspheres (I)^(a)0.68 ± 0.03 68 61 ± 14 μm^(e) Microspheres (II)^(b) 0.78 ± 0.02 78 90 ±24 μm ^(a)Prepared from 200 mg/ml polymer concentration; ^(b)Preparedfrom 300 mg/ml polymer concentration; ^(c)Average ± SD, n = 2^(d)Encapsulated efficiency = (measured loading)/(theoretical loading) ×100% ^(e)Average ± SD, n = 100.

In vitro release kinetics of rhBMP-2 from PLGA devices wascharacterized. Since rhBMP-2 has very low aqueous solubility andseverely absorbs on many surfaces, the released rhBMP-2 in PBST fromPLGA may precipitate or absorb on the surface of the container. Tominimize the loss of the releases rhBMP-2, the same release medium wasused as in the rhbFGF release study for in vitro release, i.e., 1% BSAand 10 μg/ml of heparin were combined with PBST medium.

The in vitro release profile of rhBMP-2 from PLGA cylindrical implantscontaining 0.25% rhBMP-2, 15% BSA, and 3% Mg(OH)₂ was studied. The datashowed that rhBMP-2 did not exhibit a large burst effect compared torhbFGF or BSA release from 15% BSA/3% Mg(OH)₂/PLGA millicylinders, whichmay be due to lower aqueous solubility of rhBMP-2. Over the 28 daysrelease study, rhBMP-2 was released continuously and slowly from thedevices. Since the curve of cumulative rhBMP-2 release percentage versusthe square root of release time exhibited a high linearity (R²=0.985),rhBMP-2 release from the cylindrical implants was likely diffusioncontrolled. At the end of the release study, the remaining rhBMP-2 wasextracted and quantified by the BIAcore immunoassay. The total recoveryof rhBMP-2 based on the measurement of both cumulative released rhBMP-2and remaining rhBMP-2 was above 80% (Table 9), which indicates that mostof encapsulated rhBMP-2 retained immunoreactivity.

TABLE 9 Recovery of rhBMP-2 from PLGA delivery devices. Released rhBMP-2Remaining over 28 d, % rhBMP-2, % Recover, %^(a) Cylindrical implants 55.0 ± 0.5^(b) 25.2 ± 1.0 80.2 ± 1.4 Microspheres (I) 68.7 ± 0.7 35.2 ±1.5  104 ± 1.0 Microspheres (II) 26.5 ± 1.6 21.3 ± 1.4 47.8 ± 2.7^(a)Recovery = total released percentage over 28 d + remainingpercentage ^(b)Average ± SEM, n = 3

The in vitro release profiles of rhBMP-2 from PLGA microspheres preparedfrom different polymer concentrations were studied. As expected, therelease rate of rhBMP-2 from PLGA microspheres prepared form 200 mg/mlpolymer concentration was much faster than that from 300 mg/ml polymerconcentration. Both can continuously release immunoreactive rhBMP-2 over21 days. The big burst effect for Formulation I was likely caused by thevery porous structure of these PLGA microspheres. At the end of therelease study, the extracted rhBMP-2 from PLGA microspheres wasquantified with the BIAcore immunoassay. As shown in Table 8, the fullrecovery of rhBMP-2 from Formulation I was obtainable while below 50%recovery from Formulation II, which indicated that Formulation I is morestable than Formulation II. The loss of immunoreactive rhBMP-2 inFormulation II may be due to acidic microclimate, because more acidicspecies are expected to accumulate inside the denser microspheres due tolower permeability.

Example 7 Preparation and Study of tPA/PLGA Microspheres

Mg(OH)₂ was previously used to neutralized PLGA implants to delivertherapeutic proteins, such as basic fibroblasts factor (bFGF) and bonemorphogenetic protein (BMP-2). Here, by using this rationale, tPA wassuccessfully encapsulated into PLGA implants. tPA, a protein with a MWof ˜60 K Da is a tissue type endogenous serine protease involved inthrombi dissolution. The FDA has approved the use of recombinant tPA inthe treatment of myocardial infarction. Controlled release systems forlocal delivery was developed by using hydrogel to control wound healing.A multi-drug controlled release implant with tPA encapsulated was alsotested for the intraocular management of proliferative vitroretinopathy(PVR). Here, 10% tPA powder was encapsulated as received (2% tPA, 75%arginine, 22% phosphoric acid, and 1% polysorbate 80) with or without 3%Mg(OH)₂ into PLGA millicylinders. Arginine hydrochloride and BSA wereadded in the release medium to improve the stability of released tPA.The release profile and active residue of tPA after release wasevaluated by activity analysis. The data showed that, with Mg(OH)₂encapsulated, the one month release of tPA was increased from 77.1±2.6%to 98.0±0.2% and the recovery (released part+active residue) wasincreased from 82.7±2.5% to 100.1±1.4% respectively. As far as is known,only very few protein formulations have shown continuous and completerelease over one month period without losing activity.

Example 8 Materials and Methods Reagents

Poly(D,L-lactide-co-glycolide) 50/50 with inherent viscosity of 0.20,0.63, and 0.64 dl/g in hexafluoroisopropanol was from BPI (Birmingham,Ala.). Recombinant human bFGF and BMP-2 were supplied by Scios, Inc.(Mountain View, Calif.) and Orthogene (Fremont, Calif.), respectively.Bovine serum albumin (A-3059, Lot 32HO463) and heparin (H-3393, Lot86HO454) were from Sigma Chemical Co. (St. Louis, Mo.). Fine Mg(OH)₂,MgCO₃, and Ca(OH)₂ (<5 μm) powders were obtained from Aldrich ChemicalCo. (Milwaukee, Wis.). ZnCO₃ powder (<5 μm) was from ICN BiomedicalsInc. (Aurora, Ohio). Reagents used in cell culture were from GIBCO BRLproducts (Life Technologies Inc., Md.). All other chemicals were ofanalytical grade or purer and purchased from commercial suppliers.

Preparation of PLGA Millicylinders

A uniform suspension of sieved BSA (<90 4 m) with or without Mg(OH)₂ inacetone-PLGA 50/50 (0.63 dl/g) solution (50% w/w) was loaded in asyringe and extruded into silicone tubing (I.D.=0.8 mm) at ˜0.1 ml/min.The solvent-extruded suspension was dried at room temperature (24 h) andthen dried in a vacuum oven at 45° C. (24 h). For preparation of bFGFmillicylinders, bFGF was combined with heparin, sucrose and BSAadditives at a weight ratio (additive bFGF) of 1, 180, and 1000,respectively, in 0.5 mM EDTA and 10 mM sodium phosphate buffer (pH 7.4).The solution was lyophilized for 2 days at room temperature using aLabconco Freeze Dry System (Kansas City, Mo.) to a fine powder with 4%moisture [determined by a Karl Fisher Titrator (Model DL 18,Mettler-Toledo Inc., N.J.)] and sieved before suspension in PLGA-acetoneand extrusion. For preparation of BMP-22 millicylinders, solutions ofBMP-2 combined with BSA or gum arabic (Sigma, St. Louis, Mo.) weresimilarly lyophilized and sieved. The millicylinders; were prepared inthe same way as for BSA/PLGA millicylinders. All the preparations had aloading efficiency invariably between 85% and 95%. Encapsulated proteinswere isolated from the polymer for protein assays according to theEvaluation of BSA Aggregation section.

Preparation of PLGA Microspheres

BSA was encapsulated into PLGA microspheres similar to a methoddescribed by Cohen et al. For preparation of PLGA50/50 (0.64 dl/g)microspheres, 100 μl of 150 mg/ml BSA in 10 mM phosphate buffer (pH 7.4)was added to 1 ml of 30% PLGA-CH₂Cl₂ solution with or without basicsalts. The mixture was homogenized at 10,000 rpm for 1 min on an icebath, and then immediately transferred to a 2% polyvinyl alcohol (PVA)aqueous solution. The water-in-oil-in-water emulsion was formed byvortexing the mixture for 20 s. The particles were hardened in 100 ml of0.5% PVA solution at room temperature for 3 h. The microspheres werecollected by centrifugation, washed with water, and then lyophilized toform a dry powder. For PLGA (0.20 dl/g) microspheres, 70% polymerconcentration was used. For both formulations, the particles werespherical with an average particle size between 60 and 70 μm (SD=20,N=100) determined with a microscope. The BSA loading was ˜4% with anencapsulation efficiency between 70% and 80%.

Evaluation of BSA, bFGF, and BMP-2 Release from the Polymer

Release of BSA was carried out in phosphate buffered saline/0.02% Tween80® (PBST). Millicylinders (10×0.8 mm, ˜10 mg) or microspheres (˜20 mg)were placed in polypropylene tubes containing the release medium (0.5ml) and incubated at 37° C. under mild agitation. At pre-selected times,the buffer was removed after centrifugation for analysis and replacedwith new medium. The protein content in release samples was y determinedby using a modified Bradford assay (Coomassie plus protein assay,Pierce, Rockford, Ill.), which is also compatible with the denaturingagents (e.g., 6 M urea) and reducing agents (e.g., 10 mM dithiolthreitol(DTT)) used below. At the end of release the remaining protein contentin the devices was determined as described in the next section. Therelease of bFGF and BMP-2 millicylinders was examined similarly as forBSA except that 1% BSA, 10 μg/ml heparin and 1 mM EDTA were added to therelease medium to prevent irreversible inactivation of the protein oncereleased from the polymer.

Evaluation of BSA Aggregation

PLGA devices were incubated in PBST at 37° C. At pre-selected times, theincubated polymers were removed from PBST (millicylinders) or isolatedby centrifugation (microspheres), dried with tissue paper, and dissolvedin acetone. After centrifugation and removal of the acetone polymersolution, the remaining BSA pellet was reconstituted in PBST. The BSAsolution was then incubated at 37° C. overnight before determining thesoluble protein fraction in the polymer; this gave a measure of thewater-soluble protein encapsulated (also used for protein loadingmeasurement). Any aggregate was collected by centrifugation andincubated (37° C. for 30 min) in denaturing solvent (PBST/6 M urea/1 mMEDTA); analysis of protein concentration gave the amount ofnon-covalently bonded BSA aggregates. Finally, any remaining undissolvedBSA was collected again and dissolved in reducing solvent (10 mM DTT indenaturing solvent) to determine the amount of disulfide-bondedaggregates.

Simulation of BSA Instability in the Polymer Microclimate

Three experiments were designed to simulate the potential deleteriousconditions in PLGA devices. In the pH simulation, BSA (4 mg/ml) in auniversal buffer (H₃PO₄, HAc and H₃BO₃ (40 mM each) titrated with NaOH)was lyophilized from pH 2 to 5 and incubated at 37° C. under 86%relative humidity. At various times, the protein was reconstituted inPBST and examined for the type of aggregates as described above. Toexamine the effect of water, water was directly added to lyophilized BSAat pH 2, sealed and then incubated at 37° C. for 1 week, and analyzed asabove. To examine protein adsorption, several BSA solutions (1 mg/ml) atpH values from 2 to 7 in the universal buffer were incubated for 1 weekat 37° C. with 20 mg of PLGA (0.63 dl/g) microspheres prepared bystandard solvent evaporation techniques or 20 mg of fine PLGA powder(0.20 dl/−, <100 μm). Loss of BSA content from solution was used todetermine the extent of BSA adsorption to the polymer.

Analysis of Structure and Integrity of Encapsulated BSA

Fluorescence emission spectra of BSA samples from 300 to 500 nm (240nm/min) were obtained with a Perkin-Elmer LS50B luminescencespectrometer. Far ultraviolet circular dichroism (CD) spectra (200-250nm) were recorded with a J-500A Jasco spectropolarimeter (Japan) at roomtemperature. The integrity of protein samples was determined by bothsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) andisoelectric focusing (IEF) gel electrophoresis, which were performed ona Pharmacia PhastSystem (Pharmacia Biotech.). PhastGel™ gradient 10-15gels and IEF 3-9 gels (Pharmacia Biotech.) were used for SDS-PAGE and MFanalyses, respectively. Coomassie blue staining was performed afterseparation.

Enzyme-Linked Immunosorbent Assay (ELISA) for bFGF

Immunoreactive bFGF was detected by sandwich ELISA. A 96-well plate wascoated with monoclonal anti-bFGF (Upstate Biotech. Inc., N.Y.) at 4° C.overnight. Samples or standards of bFGF containing 10 μg/ml heparin and1% BSA in PBST were added to each well and incubated at 4° C. for 24 h.After washing, polyclonal anti-bFGF (rabbit, Sigma, Mo.) was added (roomtemperature for 2 h) followed by anti-rabbit IgG-horse radish peroxidase(1:10,000, Sigma, Mo.) for another 2 h. The substrate o-phenylenediaminein the presence of H₂O₂ (Sigma Fast OPD tablet sets) was added (30 minincubation at room temperature) and the reaction was stopped by adding3M H₂SO₄. The visible product was detected at 490 nm on a plate reader(Dynatech MR7000).

BIAcore Immunoassay for BMP-2

The immunoreactive BMP-2 was quantified by an immunoassay using BIAcore2000 biosensor (Pharmacia, Uppsala, Sweden). First, a monoclonalantibody of BMP-2 (Orthogene, Fremont, Calif.) was immobilized onto aCM-5 sensor chip surface using the amine coupling kit (Pharmacia,Uppsala, Sweden). BMP-2 in the release medium was assayed over theimmobilized antibody surface. Sample volume was 30 μl and HEPES-bufferedsaline (HBS) from Pharmacia was used as the mobile phase with a flowrate of 10 μl/min. The surface was regenerated by an injection of 10 μlof 10 mM HCl (pH 2) after each injection of BMP-2 solution. The antibodyon the chip surface remained stable for more than 1 month. BMP-2 sampleswere analyzed based on the standard curve ranging from 50 to 1600 ng/mlBMP-2.

Bioassay for bFGF

The biological activity of bFGF was determined by a cell proliferationassay. Balb/c 3T3 fibroblasts (25,000/well, CCL-163, ATCQ in Dulbecco'smodified Eagle's medium (DMEM) containing 10% bovine calf serum, 50 U/mlstreptomycin and 50 μg/ml penicillin were seeded (200 μl/well) on96-well plates. The cells were grown to confluence for one week withoutchanging the medium. On day 7, bFGF samples or standards (10 μl) in therelease medium were added and followed 20 h later by addition of 1 μCiof ³H-thymidine (6.7 Ci/mmol, DuPont/NEN® Research Products, Boston,Mass.) per well. After another 6-8 h, the cells were collected on filterpaper by using a PHD TM cell harvester (Cambridge Technology Inc.). Theharvested cells were resuspended in 3 ml of scintillation cocktail 3a7OB(Research Products International Corp., Ill.) and counted (BeckmanLS1701 scintillation counter).

Results

Application of the Stabilization Strategy for bFGF Delivery

Highly acidic pH and intermediate moisture content in the polymermicroclimate are known to be deleterious conditions for most proteins.Therefore, neutralization of polymer microclimate pH should improve thestability of other encapsulated proteins. To test this hypothesis, weselected recombinant human basic fibroblast growth factor (bFGF), whichis undergoing clinical trials. Most commonly known for its potentability to promote anglogenesis, bFGF is a mitogen for a number ofmesoderm- and neuroectoderm-derived cells such as fibroblasts,endothelial cells, smooth muscle cells, osteoblasts, and melanocytes. Itis currently being tested for wound healing, osteogenesis, and diabeticulcers. Like most therapeutic proteins, its in vivo serum half-life isvery short (<3 min). Any prolonged treatment (e.g., osteogenesis) couldpotentially benefit from the development of controlled releaseformulations, but bFGF has been difficult to encapsulate inbiodegradable polymers. Polymer formulations that deliver bioactive bFGFhave been either too short-acting (e.g., because naturally derivedpolymers were used) or have been prepared from nondegradable materials,which require implant removal and therefore are undesirable in manyclinical situations.

Before encapsulating bFGF, we considered several sources of irreversibleinactivation of bFGF known to occur at neutral pH and physiologicaltemperature. This protein belongs to a family of heparin-binding growthfactors, and in the absence of heparin (or an equivalentglycosaminoglycan), it loses activity very rapidly in the presence ofelevated temperature, an acidic pH, or proteolytic enzymes. Like manyproteins, bFGF adheres avidly to glass and plastic surfaces. Disulfideexchange of bFGF has also been reported in the presence of trace metals(e.g., those remaining from the polymerization of polymer). Based on thestability profile of bFGF and our experience with BSA, we selected fiveadditives for encapsulation of bFGF. The combination of 3% Mg(OH)₂ and15% BSA were suitable for neutralization of the acidic microclimate. Thepresence of BSA at high concentration may also be useful to inhibitadsorption of bFGF to PLGA. We added heparin at a weight ratio of 1:1(heparin to bFGF) to enhance bFGF stability, and EDTA to chelate traceof heavy metals. Finally, sucrose was kept in the solid bFGF (asreceived from the supplier) to retain the bFGF structure in the solidstate.

When a small amount of bFGF was encapsulated to the Mg(OH)₂/BSA/PLGAmillicylinders (˜0.0025%), the growth factor was released in a fashionsimilar to that observed for BSA. Over 28 days, 71% of bFGF was detectedby ELISA in the release medium and 21% remained in the polymer fraction(Table 12). This accounts for about ˜92% of the initially encapsulatedbFGF. It is important to note that when the millicylinders did notcontain both heparin and the Mg(OH)₂/BSA combination, bFGF lostimmunoreactivity. For example, if heparin was removed from the stableformulation, only 2% bFGF was released over one month with noimmunoreactive bFGF in the residual fraction (Table 12). Similarly, if20% arabic gum was substituted for 3% Mg(OH)₂/15% BSA (a 0% Mg(OH)₂/15%BSA control could not be performed because of BSA aggregation), no bFGFwas observed in the release medium after 4 days and only 38% wasaccounted for in both the release and residual fraction.

To increase the capacity of the polymer to deliver bFGF, we increasedthe bFGF loading to 0.01% and the sucrose loading to 21.3%. The bFGFrelease initially is much slower and later exhibits a linear releaseprofile up to 4 weeks. The release of BSA from the same preparation inPBST was similarly retarded. This indicates that sucrose can be used toslow down the release of both BSA and bFGF from the polymer, probably byincreasing the viscosity of the aqueous pores in the polymer.

The release kinetics of bFGF demonstrated that soluble bFGF is releasedcontinuously with BSA. However, immunoreactive bFGF does not guaranteebioactivity. To test the bioactivity of bFGF released from PLGA, weexamined the bFGF samples in the release and residual fraction by theability of the growth factor to induce cell proliferation (as indicatedby ³H-thymidine incorporation). The encapsulation procedure did notaffect the biological activity of bFGF. Some small inactivationapparently occurred during the release experiment, but 65-85% of bFGFwas bioactive over the entire release interval, confirming that themajority of immunoreactive bFGF was biologically active. Thus, byneutralizing the acidic microclimate in PLGA, we have prepared aninjectable PLGA device that delivers bioactive bFGF for more than onemonth.

Generality of the Use of Basic Salts to Improve Stability of ProteinsEncapsulated in PLGA

To further test the generality of the basic additive stabilizationapproach, we encapsulated another important growth factor, bonemorphogenetic protein-2 (BMP-2), in PLGA millicylinders and formulatedBSA in PLGA microspheres. BMP-2, which has significant homology withtransforming growth factor-β, can effectively induce bone regenerationat extraskeletal sites when implanted in a suitable carrier such asinactivated collagenous bone matrix. This fascinating feature makesBMP-2/carrier systems commonly studied alternatives to bone grafting. Ithas been suggested that a central problem in the application of BMP-2for bone regeneration is the inability to slowly release the activeprotein homogeneously throughout the site of desired bone formation.PLGA is a logical choice to overcome the difficulties with the BMP-2carriers.

Following the same approach as for the bFGF formulation, we used the 3%Mg(OH)₂/15% BSA combination to neutralize the acidic microclimate pH inPLGA millicylinders containing BMP-2 (0.25% loading). In addition, weperformed a second positive control using the protein substitute, gumarabic (i.e., 3% Mg(OH)₂/15% gum arabic/0.25% BMP-2). In both cases, acontrolled release of protein was observed over 28 days (data not shown)resulting in a recovery (released+soluble residue fraction) of >80%(Table 12). In contrast, when the base was removed from the formulation(18% gum arabic), only 30% protein was recovered by the immunoassay.Therefore, the stability characteristic of BMP-2 in PLGA millicylinderswas very similar to that of bFGF (although additional stabilizers wereused for bFGF) with and without the addition of the basic additive. Forboth growth factors, in the absence of basic additive, the protein wasreleased mostly on the first day and only a small amount of proteincould be recovered from the polymer after 28 days release. In thepresence of the basic salt, both proteins were continuously releasedwith >80% recoverable in the 28-day experiment. Moreover, the in vivobiological activities (angiogenesis in nude mice for the bFGF andosteogenesis in rats for the BMP-2) of the BSA/Mg(OH)₂-containingformulations were also confirmed when the devices were implantedsubcutaneously (data not shown).

A final important consideration is the applicability of the basicadditives for, protein stabilization in PLGA microspheres. Microsphereshave several advantages compared to millicylinders, particularly toreduce pain of injections and to simplify administration. To test ourapproach in microspheres, we examined whether encapsulated BSA undergoesthe acid-induced mechanism of instability and if so, whether the basicadditive approach is effective in preventing it. As seen in Table 3, BSAalso forms non-covalent aggregates (˜25-75%) when encapsulated PLGAmicrospheres, indicating that an acidic microclimate also develops inPLGA microspheres prepared by the solvent evaporation method (we notethat in some control experiments, we also observed disulfide-bondedaggregates of BSA, but in every case, this mechanisms was secondary(<5%) to the acid-induced non-covalent mechanism). PLGA 50/50microspheres have been shown recently to form a highly acidicmicroclimate when prepared by this commonly used technique, which isconsistent with the BSA instability mechanism.

We note that it has been suggested that BSA becomes unstable in PLGAmicrospheres primarily by protein adsorption to the polymer. Thisconclusion was strongly weighed on the ability of SIDS to causeliberation of previously unreleasable BSA from the polymer. We remarkthat the SDS buffer we used in the SDS-PAGE dissolves the noncovalentaggregates formed in the polymer. This solubilization effect may explainthe reported release of sequestered BSA from the polymer caused by thesurfactant. Therefore, we conclude that protein adsorption, consistentwith our simulations described earlier, is not the predominant source ofinstability of BSA in PLGA microspheres.

Whereas the mechanism of BSA instability in microspheres was similar tothat observed in millicylinders, the co-encapsulation of Mg(OH)₂ wasonly marginally successful to inhibit BSA aggregation in microspheres.For example, the soluble fraction of BSA was increased from 17-25% (nobase) to 39%-72% (with Mg(OH)₂). This modest increase in stability ofBSA afforded by Mg(OH)₂ and previous microscopy studies from our groupillustrating a heterogeneous pH distribution in Mg(OH)₂/PLGAmicrospheres (no protein) suggests that the basic additive inmicrospheres could not diffuse to all the acidic protein pores in thepolymer. This is likely due to lower protein loading used inmicrospheres compared to that in millicylinders, which decreases thenumber of pores in the polymer. To overcome this problem, we turned toanother basic salt, MgCO₃, which has an equivalent basicity to Mg(OH)₂,but has a ˜10-fold higher solubility to facilitate diffusion of the basein the polymer pores. The more soluble carbonate salt inhibitedaggregation of BSA similarly to the inhibition attained inmillicylinders by Mg(OH)₂. For the medium molecular weight PLGA (0.64dl/g), the aggregation was held to just 13% over 28 days with 89%recovery (Table 12). Remarkably, co-encapsulation of MgCO₃ in the low MWPLGA (0.20 dl/g) resulted in reduction of BSA aggregation to just 1.5%over 51 days with 94% recovery (Table 12). This latter preparationcontrolled the release of BSA slowly and continuously over the entireexperiment after a 32% burst (data not shown).

In closing, by elucidating the deleterious conditions and mechanisms ofinstability of BSA in PLGA delivery systems, we have been able to devisea rational procedure for stabilization of BSA. This approach was alsoconfirmed for therapeutic growth factor encapsulated in millicylindersand BSA in microspheres. Our data strongly suggest that poorlywater-soluble basic salts such as Mg(OH)₂ can be used to neutralize thepolymer microclimate pH to levels necessary to retain the structure andbiological activity of acid-labile proteins encapsulated in PLGAdelivery systems.

Example 9 Stabilization of Protein in PLA-PEG Blended Microspheres

A blend of: slowly degrading poly(D,L-lactide) (PLA), to reduce theproduction of acidic species during protein release; and water-solublepoly(ethylene glycol) (PEG), to increase diffusion of BSA and polymerdegradation products, were used to modify the microsphere microclimateand protein release behavior. PLA has a much slower degradation ratethan PLGA 50/50 due to its higher hydrophobicity and the sterichindrance for the water attack of ester bond introduced by the methylgroup of lactic acid. Slow degradation of PLA results in less productionof acidic species, presumably providing a more neutral microclimate forencapsulated proteins during early incubation. However, slow degradationof PLA will also cause slow and discontinuous release of proteinantigens and a gradual acid build-up. In addition, its stronghydrophobicity has been suggested to possibly denature proteins.Therefore, the second component, relatively more hydrophilic PEG, isintroduced into PLA to adjust the microsphere hydrophobicity andpermeability. PEG is nontoxic and soluble in numerous organic solventsand water. During release, PEG can lie soluble in the release medium,resulting in the formation of swollen structure with high water contentin the polymer blend. This swollen polymer structure is expected toincrease exchange of polymer degradation products with the surroundingmedium, minimizing the risk of acid-induced protein degradation.Moreover, before excessive PLA degradation occurs, aqueous pores formedby PEG dissolution are expected to increase diffusion of theencapsulated protein, providing continuous protein release.

The PLA-PEG microspheres studied here were prepared by oil-in-oilemulsion and solvent extraction (O/O) method, instead of the mostcommonly used water-in-oil-in-water double emulsion and solventevaporation (W/O/W) method. The former approach generally results inhigh protein entrapment levels and superior protein stability due to theabsence of water. A model protein antigen, bovine serum albumin (BSA),was selected and encapsulated in the polymer blend.

Materials and Methods Chemicals

Poly(D,L-lactide) with inherent viscosity of 1.07 dl/g in CHCl₃ was fromBPI (Birmingham, Ala.). Polyethylene glycol) with molecular weight10,000 and 35,000 was obtained from Aldrich Chem, Co. (Milwaukee, Wis.)and Fluka, respectively, Bovine serum albumin: (A-3059, Lot 32H0463) waspurchased from Sigma Chemical Co. (St. Louis, Mo.). Protein molecularweight and pI standards for electrophoresis were from Pharmacia LKB(Piscataway, N.J.). All other biochemicals and chemicals were ofanalytical grade or purer and obtained from commercial suppliers.

Microsphere Preparation

The polymeric microspheres were prepared by an anhydrous O/O method.First, PLA and PEG at various weight ratios were co-dissolved inacetonitrile at a total polymer concentration of 20% (w/v). Sieved BSA(<20 μm) was suspended in acetonitrile-polymer solution and homogenizedat 15,000 rpm in an ice bath, Then the antigen suspension was addeddrop-wise into the continuous phase (cottonseed oil containing 1.6%(W/V) span 85) stirred at 750 rpm with an overhead stirrer. After 5 hr,petroleum ether (b.p. 50-110° C.) was poured into the cottonseed oilbath to extract the remaining acetonitrile from the polymer. After anadditional 15 min of stirring, the microspheres were filtered, washedwith 250 ml of petroleum ether and lyophilized.

Microsphere Characterization Morphology and Particle Size Determination

The microspheres were coated with gold-palladium by using PELCO MODEL 3SPUTTER COATER 91000. Surface morphology of the microspheres wasexamined by a Philips XL Scanning Electron Microscope. Particle size wasestimated by averaging diameters of 50 microspheres.

Polymer Composition Analysis by IR

The composition of microspheres prepared from different blends of PLAand PEG was analyzed by infrared spectroscopy. A Nicolet protege 460 wasused to obtain the spectra (32 scans per sample, over 600-4000 cm⁻¹) forthe samples. A series of PLA and PEG physical mixtures with differentweight ratios was used to make a calibration curve. Samples weredissolved in chloroform and casted into a sodium chloride cell. Thecomposition of the microparticles was estimated by comparing peak heightratios corresponding to the carbonyl (C═O) band of PLA at 1757 cm⁻¹ andthe CH₂ band at 2876 cm⁻¹ due to the PEG component, and assuming anegligible content of span 85 surfactant in microspheres.

Polymer Phase Behavior Analysis by DSC

Samples (3-5 mg) were loaded into aluminum pans and DSC thermograms wererecorded by a Perkin-Elmer DSC 7 Differential Scanning Calorimeter.Nitrogen gas was the sweeping gas and the heating rate was 20° C./min.

Determination of Microsphere Loading

The amount of antigen encapsulated in microspheres was determined byrecovering the protein from the microspheres. First, acetone was addedto the microspheres to dissolve the polymer. The mixture was vortexed,centrifuged and then supernatant was removed. After the removal ofpolymer was repeated three times, the remaining protein pellet was airdried and reconstituted in phosphate buffer saline pH 7.4 containing0.02% Tween 80® (PBST) and protein content was determined by theCoomassie Plus method (Pierce Chem. Co., Ill.)

Evaluation of Model Antigen Release From Microspheres

Samples of 10 mg microspheres were suspended in 1 ml PBST. Thesuspension was incubated at 37° C. under mild agitation. At pre-selectedintervals, release media were removed for determination and replacedwith fresh buffer. The amount of protein released was assayed by theCoomassie Plus method (Pierce Chem. Co., Ill.). At the end of release,microspheres were collected and remaining soluble protein in themicrospheres was analyzed as described in the section Determination ofmicrosphere loading. Any insoluble protein aggregates were collected bycentrifugation and reconstituted in denaturing agent (8 M Urea or 6 MGuanidine-HCl (GnCl)). Determination of any aggregates soluble indenaturing agent gave the amount of non-covalently bonded aggregates.With the further addition of reducing agent (10 mM DTT+1 mM EDTA), anydisulfide-bonded aggregates were dissolved. The total dissolved portionin denaturing and reducing agents gave the total amount of non-covalentand disulfide-bonded aggregates.

pH Change in the Release Medium During Release

The pH of the release medium was monitored by a Corning 430 pH meter(Corning Inc., N.Y.) at each sampling interval.

Water Uptake of Microspheres

After incubation at 97% relative humidity and 37° C., samples were takenout and weighed immediately. The water uptake of microspheres wasestimated by:

Water uptake(%)=(W ₁ −W ₂)/W ₂×100%

Where W₁ and W₂ are the weights of the hydrated microspheres andmicrospheres before incubation, respectively. No corrections were madefor inter-particle water content in W₁ or the water content withinlyophilized microspheres in W₂.

Structural Analysis of Encapsulated BSA

At the end of release period, the integrity of remaining BSA in thepolymer was determined by both sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and isoelectric focusing (IEF) analysis,which were performed on a Pharmacia PhastSystem (Pharmacia Biotech.)according to the file no. 110 and 100 in the Phastsystem™ User Manual,respectively. In both analyses, Coomassie staining file no. 200 wasused. The secondary structure of antigen samples was determined laymeasuring circular dichroic (CD) spectra. The spectra were taken with aJ-500A Jasco spectropolarimeter (Hachioji, Japan) at room temperature.The tertiary structure of protein samples was analyzed by measuring theintrinsic fluorescence emission spectra. Fluorescence emission spectra(300-500 nm for BSA) were obtained on a Perkin-Elmer LS50B luminescencespectrometer scanned at 240 nm/min. The excitation wavelength for BSAwas set to 295 nm. Details of these procedures were as describedpreviously.

Results Microsphere Composition and Phase Behavior Analysis

The IR spectra of blank microspheres prepared from pure PEG, pure PLA(i.v.=1.07 dl/g), and a PLA/PEG displayed a broad band at 2876 cm⁻¹ anda peak at 1757 cm⁻¹, which were assigned to the CH₂ stretching on thePEG unit and the carbonyl group (C—O) of PLA, respectively. Bothcharacteristic peaks for CH₂ and C═O appeared in the IR spectrum of theblend of PLA and PEG. By estimating the PEG content in the blend with acalibration curve generated from PLA and PEG physical mixtures withdifferent weight ratios, complete incorporation of PEG in PLA matrix bythe O/O encapsulation method was indicated.

The DSC thermograms of prepared blank microspheres show. PLA exhibited aT_(g) of roughly 53.5° C. and PEG exhibited a T_(m) of 71° C. In the PLAand PEG microsphere blend, down shifts in both T_(g) and T_(m) of 7-11°C. and 8-10° C., respectively, were observed, indicating partialmiscibility between PLA and PEG.

Microsphere Morphology

After preparation, microspheres with different weight ratios of PLA andPEG had spherical and smooth surfaces. An average size of 100 μm wasrecorded for these microsphere preparations. After 35 days ofincubation, microspheres prepared from 100% PLA remained intact with asmooth surface. With the blend of PEG, the microsphere structure stillremained intact, but a small amount of pores appeared on the PLA/PEGmicrospheres surface. With higher PEG blend, more pores became visible.In addition, the microsphere surface showed indentations, which may haveoccurred during drying of the particles before analysis. The SEM imagessuggested that the incorporation of PEG into PLA created more channelsin the microspheres, which may have increased the permeability to theencapsulated protein. In addition, the microsphere surface likelyconsisted of a PLA-rich phase, whereas the interior of microspheres waslikely PEG-rich. Otherwise, more pores created by PEG solubilizationwould be expected on the microsphere surface. The PLA-rich surfacephenomenon is possibly due to the higher hydrophobicity and longer chainof PLA, which could have caused selective PLA precipitation at thesurface during the 0/0 microsphere preparation. Further surface analysiswould be required for a definitive conclusion.

Release Kinetics and Stability of BSA in the PLA/PEG Microspheres

To investigate the effect of PEG in the PLA/PEG microspheres,microspheres with different weight ratios of PEG 10,000 to PLA wereprepared and the BSA controlled release was monitored in PBST at 37° C.Theoretical BSA loading of all these formulations was 5% andencapsulation efficiency vas invariably between 90% and 100%. When PEGcontent was less than 10% of polymer weight, similar release kinetics ofBSA from microspheres was observed and less than 45% of BSA was releasedafter a 4-week incubation. When PEG content was raised to 20%, the totalreleasable amount of protein was significantly increased to 75%. Inaddition, the effect of PEG molecular weight on protein release was alsoevaluated. BSA had almost identical release kinetics in microspheresirrespective of whether PEG 10,000 and PEG 35,000 was used (the weightratio of PEG/PLA was 20:80). When PEG 35,000 content was increased to30% in PLA/PEG microspheres, a higher burst release of BSA was observed.

The residual BSA remaining in these devices after the 4-week releaseinterval (45 days for formulation without PEG, i.e., formulation o) wasanalyzed. For the formulation o, of the original encapsulated protein,15% was still water-soluble and 25% of BSA had become water-insolubleaggregates in the residue. Most of the aggregates were soluble in adenaturing solvent (6 M urea), indicating their non-covalent character.When 5% of PEG was incorporated in PLA (formulation a), soluble BSAremaining in microspheres was increased to 30%, and the non-covalentaggregates were 41% of the original encapsulated BSA, When PEG contentwas increased to 10% (formulation b), 36% of the protein formedinsoluble aggregates, Besides non-covalent aggregates, a small fractionof disulfide-bonded aggregates (soluble in 10 mM DTT) was also formed,However, no insoluble BSA aggregates were observed in formulationscontaining more than 20% PEG.

The integrity of the soluble BSA recovered from the polymer (28-dayincubation) was further examined by SDS-PAGE. Some peptide fragmentswere observed in lanes 6 and 7 (formulations a and b), indicating mildpeptide bond hydrolysis occurred during incubation. In contrast, solubleBSA recovered from formulations containing more than 20% PEG showed avery similar band with standard BSA and no degradation product bandswere noticeable. Soluble BSA recovered from formulations c, d and e wasfurther examined by IEF. No pI alterations in BSA were observed in thesesamples. Likewise, secondary and tertiary structure of BSA was similarto standard BSA control. Hence, the structure of BSA in formulations c,d, and e was retained within the polymer for one month.

Mechanisms of BSA Stabilization in the PLA/PEG Microspheres

One-month continuous release of stable BSA from microspheres wasachieved when PEG content in the PLA/PEG blends was above 20%. Asidentified previously, an acidic microclimate and intermediate moisturelevels are the two major factors which cause non-covalent aggregationand peptide-hydrolysis of BSA in PLGA 50/50 microspheres. Does the blendof PEG with PLA improve the microclimate as we designed, i.e., byavoiding the acidic microclimate and increasing the water content tostabilize BSA encapsulated in microspheres?

To address this question, we first examined the pH change of the releasemedium when the PLA/PEG formulations were incubated at 37° C. and PEST(pH 7.4). Unlike PLGA 50/50, which showed a dramatic pH drop in therelease medium after 4-week incubation, both PLA and PLA/PEGformulations remained a relatively neutral pH (above 7) in the releasemedium over 29 days of incubation. However, a slightly lower pH in therelease medium incubated with PLA/PEG formulation than that in PLA wasobserved (−0.1-0.2 pH units difference). This result suggested that someacidic degradation products were able to diffuse out of polymer devicethrough the water channels formed by PEG in PLA/PEG formulation. Inaddition, by using a previously reported method (pH determination ofpolymer solution in the mixture of ACN and water), the pa_(H)* insideformulation d before and after 30-day incubation was determined as 6.5and 5.4, respectively, suggesting a very small accumulation of acid inthe polymer. In contrast, PLGA 50/50 microspheres were reported to reachpa_(H)* ˜3 after similar incubation time. These results demonstratedthat acid build-up was largely reduced in the PLA/PEG blend formulation.

The water content difference in formulations during release was comparedby performing a water uptake kinetics study of microspheres at 97%relative humidity. Under controlled humidity, microspheres will adsorbwater vapor and potential water uptake of different formulations duringrelease can be predicted and compared. PEG 35,000 showed a strong wateruptake. On the second day, the water content in PEG 35,000 blankmicrospheres was almost 120% of the dry microsphere weight. Uponblending PEG in the formulation, the water uptake rate was significantlyincreased. The higher the PEG content, the higher the increase in wateruptake. Microspheres containing 20% PEG had almost twice the amount ofwater uptake; relative to those with 10% of PEG in the humidenvironment. When microspheres are incubated in the release medium,higher water content in the PLA/PEG blend is expected. The presence of5% BSA did not increase water uptake rate significantly in the blend.formulation. The water uptake in the blend was likely overwhelmed by thestrong water adsorption by PEG.

The above results demonstrated that a less acidic and more hydrophilicmicroenvironment was achieved in the PLA/PEG blend. Maintenance of arelatively neutral microclimate in PLA/PEG blend formulation can beattributed to the following. First, few acidic species were producedduring early incubation due to the slow degradation of PLA. The rateconstant of PLA degradation at 37° C. in water has been reported to beroughly 0.012 day⁻¹, much slower than PLEA 50:50, 75:25, 85:15 with rateconstants of 10.55, 0.103, and 0.026 day⁻¹, respectively. In addition,prior to hydration, the polymer acid content was determined as 21 and4.2 nmol/mg for the PLGA 50/50 used in our previous study and PLA usedhere, respectively. Therefore, the total amount of acidic species in PLAshould be less than PLGA either during encapsulation or duringhydration. Second, the blend of PEG with PLA significantly increased thewater content in the formulation, which is expected to dilute the acidicspecies even further. Third, the dissolution of PEG in the releasemedium may create more water channels, thereby increasing the diffusionfor acidic species out of the polymer and for buffering species into thePLA matrix.

By the above three mechanisms, a less acidic microclimate will be formedin the PLA/PEG blend. When PEG content is less than 10% in the blendformulations, non-covalent aggregates and peptide fragments of BSA werestill observed, This is possibly due to regional acidity in the polymerwhich caused BSA degradation. Slowly produced polymer degradationproducts in certain regions can not be diluted or diffuse out of thepolymer because of insufficient water channels, resulting in regionallylow ply. With increasing amount of PEG in the blend, a relativelyneutral microclimate was gradually attained. Although slight pHdecreases within the polymer was still detected in the blend containing20% PEG, it was not significant enough to cause non-covalent aggregationand peptide-hydrolysis of BSA.

The stabilization of BSA in the PLA/PEG microspheres can also beattributed in part to the increased water content in the formulation. Itwas reported that the aggregation of BSA at acidic pH (pH=2) exhibit apronounced bell-shape with maximum aggregation corresponding to roughly1008 water/100 g dry protein. When water content increased to500%-1000%, aggregation of BSA was declined sharply. In the blendformulation containing 20% of PEG, when incubated at 97% R.H. for 1week, the water content in the microspheres is 25%. Assuming all thewater is available for BSA (BSA loading is 5%) in microspheres, thewater content of BSA is 500%. During release, the water content inmicrospheres was expected to be higher than 500%. Thus, in addition tothe minimal acid content, the aggregation of BSA was minimized by thehigh amount of imbibed water in the microenvironment.

PEG is hydrophobic in nature and it may potentially interact with thehydrophobic groups of BSA and induce BSA unfolding. It was reported thatPEG of low MW 1000 and 4000 interacts favorably with hydrophobic sideschains of human serum albumin (hSA), leading to a stabilization of theunfolded state. To test the interaction of high MW PEG with BSA, GnClunfolding curve of BSA with the addition of PEG 10,000 and PEG 35,000(the weight ratio of BSA to PEG was 1:5) was determined by fluorescencespectroscopy. Similar unfolding curves were observed in threepreparations, The conformational stability of BSA was therefore likelynot affected by the addition of PEG 10,000 and PEG 35,000 with 1:5 ratioof BSA to PEG.

CONCLUSIONS

By using the PLA/PEG blend, a one-month continuous release of BSA wasachieved with the absence of insoluble aggregates and peptidehydrolysis. This formulation can be used potentially for encapsulationof other acid-labile pharmaceuticals and vaccine antigens.

TABLE 10 Irreversible inactivation of BSA under simulated andencapsulated conditions at 37° C. Encapsulated^(a) Simulated^(b) Time to50% aggregation 12 days 7 days Aggregates soluble in >98% >94%denaturing solvent^(c) Peptide fragmentation^(d) 25, 40, 25, 40, and 55kDa and 55 kDa ^(a)15% BSA in PLGA millicylinders incubated in PBST at37° C.; ^(b)lyophilized BSA at pH 2 incubated under 86% R.H. at 37° C.;^(c)PBST containing 6 M urea and 1 mM EDTA; ^(d)from SDS-PAGE of BSAsamples treated with SDS and β-mercaptoethanol.

TABLE 11 Neutralization effect of Mg(OH)₂ on the erosion behavior of 15%BSA/PLGA millicylinders No salt 3% Mg(OH)₂ Non-covalent aggregates^(a) %65 ± 8 2.0 ± 0.4 Water uptake^(b), % 48 ± 2 106 ± 4  PLGA degradationt_(1/2) ^(c), days 16.0 25.1 PH of the medium^(d) 3.5 7.0 ^(a)extractedfrom the devices after incubation in PBST at 37° C. for 2 weeks (mean ±SEM, n = 3); ^(b)determined by weighing the wet and dry devices afterincubation in PBST at 37° C. for 1 weeks (mean ± SEM, n = 3);^(c)t_(1/2) is the time when the PLGA MW (determined by GPQ reduced tohalf of the original MW during incubation in PBST at 37° C.; ^(d)PBSTmedium containing 5 mg polymer device after incubation at 37° C. for 4weeks; No medium pH change was detected in either sample for the first 3weeks.

TABLE 12 Generality of the stabilization effect of basic salts forprotein delivery from PLGA Released Soluble Insoluble RecoveryFormulations % Residue^(f), % Residue^(g), % % BSA/PLGA No base   4.4 ±0.1^(e) 17 ± 2 68 ± 6 90 microspheres 3% Mg(OH)₂  6.9 ± 0.2 65 ± 2 26 ±1 98 (0.64 l/g)^(a) 3% MgCO₃ 17 ± 2 59 ± 1 13 ± 2 89 BSA/PLGA No base 16± 2  0.9 ± 0.1 24 ± 3  41^(h) microspheres 3% Mg(OH)₂ 37 ± 2  2.1 ± 0.130 ± 2  69^(h) (0.20 dl/g)^(b) 3% MgCO₃ 68 ± 2 24 ± 1  1.5 ± 0.2 94BFGF/PLGA 15% BSA/3% Mg(OH)₂/no heparin  1.9 ± 1.3 0  2millicylinders^(c) 20% gum arabic/heparin 32 ± 1  6 ± 3 38 15% BSA/3%MG(OH)₂/heparin 71 ± 5 21 ± 2 92 BMP-2/PLGA 15% BSA/3% Mg(OH)₂ 55 ± 1 25± 1 80 millicylinders^(d) 18% Gum arabic 24 ± 3  6 ± 1 30 15% Gumarabic/3% MG(OH)₂ 60 ± 9 23 ± 2 83 ^(a)BSA loading was −4% and therelease study was carried out for 28 days; ^(b)BSA loading was −4% andthe release study was carried out for 51 days; ^(c)bFGF loading, was−0.0025% and the release study was carried out for 28 days; ^(d)BMP-2loading was −0.25% and the release study was carried out for 28 days;^(e)All the data represent mean SEM, n 3; ^(f)Soluble in PBST (BSA) orin PBST/1% BSA/10 ~Lglml heparin for (bFGF and BMP-2); ^(g)Insoluble inPBST but soluble in 6 M urea; ^(h)Less than the Computed recovery wasobserved in the unstable preparations.

Example 10 PLGA Microspheres which Stabilize Vincristine Sulfate (VCR)

Vincristine sulfate (VCR) and vinblastine sulfate (VBL) are two vincaalkaloids that are commonly used as single agents or in combination forsystemic treatment of AIDS-KS. VCR and VBL structurally identical withexception of the group attached to the nitrogen at position 1, at whichVCR possesses a labile N-formyl group and VBL has a stable methyl group.Both drugs undergo pH-dependent degradation in aqueous solution, the pHof maximum stability is ˜2 for VBL and ˜4.5 for VCR. This exampledemonstrates that VCR becomes unstable in PLGA (50% D,L lactide content)microspheres, whereas encapsulated VBL is highly stabilized. Thisexample provides PLGA microspheres that stabilize VCR for over a month.

Materials and Methods Chemicals

Vincristine sulfate (98% purity) and vinblastine sulfate (97% purity)were obtained from Sigma (St. Louis, Mo.). PLGA with copolymer ratio ofD,L-lactide to glycolide 50:50 and inherent viscosity of 0.23 dl/g waspurchased from Birmingham Polymers (Birmingham, Ala.), Mg(OH)₂ wasobtained from Aldrich Chemical Co. (St. Louis, Mo.) and ZnCO₃ waspurchased from ICN Biopharmaceuticals (Aurora, Ohio). All other reagentsand solvents were of analytical grade or purer and purchased fromcommercial suppliers.

Microspheres Preparation

Microspheres were prepared by a standard oil-in-oil emulsion-solventextraction method. 150 mg PLGA were dissolved in 150 μl of acetonitrile(CAN) before addition of 15 μl of aqueous VCR or VBL solution (20mg/ml). In some instances, Mg(OH)₂ or ZnCO₃ at 0.5, 3, and 10% (wt.base/wt. polymer) were suspended in the polymer solution to raise themicroclimate pH inside the microspheres. The resulting solution orsuspension was added drop-wise to 25 ml of oil (95% cottonseed oil and5% Span 85 emulsifier) stirred at 500 rpm and room temperature. After2.5 h of microsphere hardening. 40 ml of petroleum ether (bp: 50 to 100°C.) were added to the emulsion to extract CAN. The emulsion was stirredfor additional 15 min, the particles were collected by filtration, andwashed 3 times with petroleum ether. The hardened microspheres wereflash-frozen with liquid nitrogen and lyophilized with a LabconcoFreezone 6 system for 1 day.

Analysis of Drugs and Their Degradation Products by HPLC

VCR and VBL were examined by high performance liquid chromatography(HPLC). The HPLC system consisted of the following: a 510 pump, a 717Plus autosampler, and a 486 UV detector (waters, Milford, Mass.). A C₁₈3.9×150 mm reverse phase column (Waters Nova-Pak) was used at a flowrate of 1 m./min. The mobile phase was composed of aqueous solution ofsodium phosphate (10 mM) and methanol 40:60 (v/v) (pH 7.0). For UVdetection, the wavelength was set to 298 nm.

Identification of VCR Degradation Product by LC-MS

For identification of VCR and its degradation products a LC/MS systemwas used. The system consisted of a Perkin-Elmer Sciex API 300triple-quadruple mass spectrometer (Thornhill, Ontario, Canada) coupledto a Schimadzu HPLC system (Columbia, Md.). The HPLC system was equippedwith an SCL-1A system controller, a LC-10A pump, a GT-104 degasser, andan SIL-10A autosampler. The separation of the parent drug and thedegradation products was performed in 10 mM ammonium formate (pH 4) andCAN (40/60 v/v) on a C₁₈ reversed phase column.

Microscopic Evaluation of Microsphere Size Distribution and Morphology

Greater than one hundred particles for each preparation were sized bysight under Zeiss Axiolab light microscope equipped with a 10× objectiveand a sizing scale bar. Scanning electron microscopy (SEM) images ofPLGA microspheres were obtained by using a Philips XL30 field emissiongun scanning electron microscope. Samples were coated with conductivegold prior to analysis.

Evaluation of VCR and Its Degradation Products During Release

Drug release from microspheres was carried out in PBS (127 mM NaCl, 3 mMKCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, pH 7.4) containing Tween 80 (0.02% w/w/)(PBST) at 37° C. under perfect sink conditions. VCR and VBL wereunstable in the release media so release kinetics was monitoredindirectly from the drug remaining in the polymer. Microspheres wereweighed and dissolved in a 50% (v/v) ACN water solution. Theprecipitated polymer and salts were spun down by brief centrifugation.An aliquot of the supernatant containing drug was removed and analyzedby HPLC.

Non-Aqueous Solvent pH Measurements

150 mg of PLGA were dissolved in 40 μl CAN and ZnCO₃, and Mg(OH)₂ wassuspended in the polymer solution at 0.5, 3, and 10% 9 w. base/wt.polymer). 15 μl of double distilled water were added to the suspensionand vortexed for 20 s to simulate microsphere preparation conditions.The undissolved salts were spun down by a brief centrifugation and thesupernatant was diluted in an CAN:H₂O mixture to make a 50 mg/ml polymerconcentration. The final solvent composition was 80:20 (v/v) CAN:H₂O.The pH was measured with a Corning Semi-Micro Combination glasspH-electrode attached to a Corning pH meter (VWR scientific, Pa.). Asdescribed previously, the actual proton activity in the organic solutionmixture (a) was calculated from the pH meter reading (pH) by pa=pH−δ,where δ is a correction coefficient which equals 0.95 for an ACN:H₂O80:20 (v/v) mixture.

Results Degradation of VCR Encapsulated in PLGA Microspheres

Microspheres containing 0.22% (w/w) drug were obtained by an oil-in-oilemulsion-solvent extraction technique. The encapsulation efficiency was˜91% (Table 13, Protocol A). Microspheres were spherical in shape withthe mean particle size of 46 μm.

All the encapsulated vincristine was originally preserved in its nativeform following encapsulation. During microsphere incubation, the drugdegraded rapidly inside the particles. The appearance of a majordegradation product was observed in the chromatogram (peak II). Only 23%of drug remained in its native form after 14 days of the incubation.Curve fitting assuming pseudo-first order kinetics for the degradationof encapsulated VCR gave a rate constant of k=1.07 10⁻⁶x⁻¹ and t₁₂=7.5days at 37° C.

In order to improve drug stability in the formulation, the followingmethodology was used: (a) identification of the degradation product, (b)elucidation of the cause and mechanism of VCR degradation in the PLGA,and (c) stabilization of VCR in PLGA microspheres by inhibiting orbypassing the cause and mechanism of VCR degradation.

Identification of the VCR Degradation Product

The degradation product was more hydrophobic relative to the parent drugsince its retention time (peak II at 7.6 min) was longer than theretention time of VCR (peak I at 5.5 min). LC-MS analysis revealed themain molecular peaks of 797.5 Da for the degradation product and 825.5Da for VCR. The difference of 28 Da was likely due to the loss ofN-formyl group at the position 1. Formation of the deformyl derivativeof VCR was reported previously by Sethi et al. and is favorable atacidic pH. The retention time of the degradation product formed in PLGAmicrospheres also corresponds to the retention time of VCR degradationproduct formed in solutions pH 1.5.

TABLE 13 Characterization of Microspheres Protocol Drug Base Baseloading, Drug loading Encapsulation Particle Yield, code added added %(w/w) % (w/w)^(a) efficiency, %^(a) size, μm^(b) % A VCR — — 0.22 ± 0.0191 ± 1 46 ± 3 89 B VBL — — 0.18 ± 0.01 88 ± 3 50 ± 2 93 C VCR Mg(OH)₂0.5 0.15 ± 0.02 76 ± 8 42 ± 3 91 D VCR Mg(OH)₂ 3 0.272 ± 0.01  98 ± 1 59± 4 87 E VCR Mg(OH)₂ 10 0.18 ± 0.01 94 ± 1 50 ± 3 94 F VCR ZnCO₃ 3 0.15± 0.01 82 ± 3 52 ± 3 89 G VCR ZnCO₃ 10 0.19 ± 0.02 87 ± 5 43 ± 5 92^(a)N = 3 ± SD. ^(b)N = 100 ± SEM.

Co-Encapsulation of Mg(OH)₂ in PLGA Microspheres Stabilizes VCR

The encapsulation of insoluble bases in PLGA microspheres causes anincrease in the microclimate pH and an inhibition of acid-inducedinstability of encapsulated proteins. To inhibit acidic degradation ofVCR, Mg(OH)₂ was co-encapsulated in PLGA microspheres at 0.5, 3 and 10%(wt. base/wt. Polymer) loading. The addition of base did not change thespherical appearance of microspheres, although the particle surface athigh base content became less smooth due to protruding base particles. Amicrosphere particle size of ˜50 μm, a loading of ˜0.2%, and anencapsulated efficiency in a range of 76 to 98% were obtained (Table 13,Protocols C-E).

The acidic degradation of VCR was fully inhibited by addition of 3 and10% of Mg(OH)₂ the deformyl degradation product appeared after 2 weeksof incubation. This can be attributed to non-homogeneity of microclimateneutralization by Mg(OH)₂ and/or an insufficient supply of base for theneutralization of acidic groups formed as PLGA hydrolysis proceeded.

Despite VCR stabilization during release, the addition of Mg(OH)₂induced the appearance of a second degradation product formed duringmicrosphere preparation (peak III. The degradation product was morehydrophilic with a retention time of 2.6 min compared to 5.5 min forVCR. The retention time of peak III is consistent with that of the VCRdegradation product formed in solution in pH 7.3 in the study by Vendriget al. Roughly 12% of the drug was degraded during the preparation ofthe microspheres containing 3 and 10% Mg(OH)₂. No further formation ofthe basic degradation product was observed during microsphereincubation. It is probable that VCR is either exposed to a higher pH oris more reactive in the polymer-base solutions during microspherepreparation than in the polymer microclimate during incubation.

Substitution of Mg(OH)₂ with ZnCO₃ Inhibits Alkaline Degradation

To inhibit formation of the basic degradation product a weaker base,ZnCO₃ and 9.8 for Mg(OH)₂. The hydronium ion activities in non-aqueoussolvents (pa_) of the polymer solutions with and without bases weremeasured to evaluate the conditions affecting VCR stability duringmicrospere preparation (Table 14). The pa_of PLGA solution containing noadditives was low at 3.9. This value increased with addition of 0.5, 3,and 10% of Mg(OH)₂ to 4.8, 6.1 and 7.3, respectively. The addition ofZnCO₃ also increased pa_but to a lesser extent than the addition ofMg(OH)₂ on a weight basis.

The substitution of Mg(OH)₂ with ZnCO₃ did not change the physicalcharacteristics of the microspheres. Sperical microspheres with ˜0.17%drug loading, 85% encapsulation efficiency, and the ˜48 μm particle sizewere obtained (Table 13, Protocols F-G). However, only 3% of VCRconverted to the basic product during microsphere preparation with ZnCO₃compared to 12% with Mg(OH)₂. The acid-catalyzed VCR degradation wasinhibited resulting in 97% of the drug remaining intact after 3 weeksand 92% intact after 4 weeks. Hence, the substitution of Mg(OH)₂ withZnCO₃ further improved the stability of encapsulated VCR in PLGAmicrospheres.

Drug Release Kinetics

Drugs were released in sustained manner from all the formulations. VBLmicrospheres released drug nearly linearly for 4 weeks (66% ofencapsulated drug released). The VCR formulations contained 3 and 10%ZnCO₃ and released 56 and 31% of the stable drug, respectively, at theend of incubation period. VCR was released faster from the formulationcontaining more ZnCO₃, probably because the co-encapsulation of thisbased increased polymer water content leading to a faster drugtransport. The water content of PLGA usually increases withco-encapsulation of basic additives and microclimate neutralization. Forexample, the water content was reported to increase 2.5 and 4 times byco-encapsulation of 3% Mg(OH)₂ and 3% ZnCO₃, respectively, for PLGAmillicylinders containing 15% protein. Hence, we expected to observe anincrease in VCR release rate from the formulations containing basescompared formulations containing bases compared to the formulationswithout base. However, just the opposite was observed as less drug wasreleased after 28 days from the microspheres containing either 3-10%Mg(OH)₂ or 3% ZnCO₃ compared to microspheres without additives. Apossible explanation is that the solubility of weakly basic drugdecreased in the neutralized microclimate (VCR) has pK_(a)s of 5 and7.4). In addition, the positively charge drug may have interacted withthe negatively charged polymer end-groups, which become ionized in theneutralized microenvironment.

TABLE 14 Neutralization of PLGA Solutions with Basic Salts Base Baseloading, added % (w/w) pa_(——) — — 3.9 ± 0.1 Mg(OH)₂ 0.5 4.8 ± 0.3Mg(OH)₂ 3 6.1 ± 0.4 Mg(OH)₂ 10 7.3 ± 0.2 ZnCO₃ 3 5.0 ± 0.1 ZnCO₃ 10 6.4± 0.3 ^(a)N = 5 ± SD.

1. A method of preparing a biodegradable polymeric delivery system fordelivering a biologically active agent to a subject, comprising: a)forming a polymer solution comprising a solvent and apoly(lactide-co-glycolide) (PLGA) polymer which comprises from 50 to100% lactide or lactic acid and from 50% to 0% glycolide or glycolicacid, wherein said lactide or lactic acid is selected from the groupconsisting of the L isomer, the D isomer, or a D,L racemic mixture; b)blending a pore-forming agent selected from the group consisting ofpolyethylene glycol (PEG), poloxamer, and combinations thereof, with theresulting polymer solution of (a) at a level of from 10% to 30% (w/w,pore-forming agent/polymer); c) dispersing the biologically active agentin the resulting polymer solution of (b); and d) solidifying the polymerfrom the resulting polymer solution of (c), wherein the method resultsin the formation of a polymeric system whose microclimate maintains,without the need for added bases or buffers, a pH of greater than 3during biodegradation for a period of at least 4 weeks.
 2. The method ofclaim 1, wherein the organic solvent is selected from the groupconsisting of acetone, methylene chloride, acetonitrile, chloroform, andcombinations thereof.
 3. The method of claim 1, wherein the amount ofpore-forming agent blended with the resulting polymer solution of (a) isfrom 10 to 20% (w/w, pore-forming agent/polymer).
 4. The method of claim1, wherein the amount of pore-forming agent blended with the resultingpolymer solution of (a) is from 20 to 30% (w/w, pore-formingagent/polymer).
 5. The method of claim 1, wherein PEG has a molecularweight of from 500 to 30,000.
 6. The method of claim 1, wherein PEG hasa molecular weight of from 4000 to 10,000.
 7. The method of claim 1,wherein the pore-forming agent is a water-soluble poloxamer having amolecular weight of from 500 to 30,000.
 8. The method of claim 1,wherein the poloxamer has a molecular weight of from 4000 to 10,000. 9.The method of claim 1, wherein the polymer comprises 100% lactic acid orlactide and wherein the amount of PEG blended with the resulting polymersolution of (a) is from 10 to 20% (w/w, PEG/polymer).
 10. The method ofclaim 1, wherein the microclimate of the resulting biodegradablepolymeric system maintains a pH of from 3 to 5 during biodegradation.